Genome-wide association study of bacterial blight resistance in soybean.

Bacterial blight caused by Pseudomonas syringae pv. glycines (Psg) is a widespread foliar disease. Although four Resistance to Pseudomonas syringae pv. glycinea (Rpg) 1 ~ 4 (Rpg1~4) genes that have been observed to segregate in a Mendelian pattern have been reported to confer resistance to Psg in soybean, the genetic basis of quantitative resistance to bacterial blight in soybean remains unclear. In the present study, the Psg resistance of two soybean association panels consisting of 573 and 213 lines, respectively, were phenotyped in multiple environments in 2014 - 2016. Genome-wide association study (GWAS) were performed using 2 models FarmCPU and BLINK to identify Psg resistance loci. A total of 40 soybean varieties with high level of Psg resistance were identified, and 14 quantitative trait loci (QTLs) were detected on 12 soybean chromosomes. These QTLs were identified for the first time. The majority of the QTLs were only detected in one or the other association panels, while qRPG-18-1 was detected in both association panels for at least one growing season. A total of 46 candidate Psg resistance genes were identified from the qRpg_13_1, qRPG-15-1, and qRPG-18-1 loci based on gene function annotation. In addition, we found the genomic region covering rpg1-b and rpg1-r harbored the synteny with a genomic region on chromosome 15, and identified 16 nucleotide binding site - leucine-rich repeat (NBS-LRR) genes as the candidate Psg resistance genes from the synteny blocks. This study provides new information for dissecting the genetic control of Psg resistance in soybean.

An Improved Method for Detecting Crane Wheel-Rail Faults Based on YOLOv8 and the Swin Transformer.

In the realm of special equipment, significant advancements have been achieved in fault detection. Nonetheless, faults originating in the equipment manifest with diverse morphological characteristics and varying scales. Certain faults necessitate the extrapolation from global information owing to their occurrence in localized areas. Simultaneously, the intricacies of the inspection area's background easily interfere with the intelligent detection processes. Hence, a refined YOLOv8 algorithm leveraging the Swin Transformer is proposed, tailored for detecting faults in special equipment. The Swin Transformer serves as the foundational network of the YOLOv8 framework, amplifying its capability to concentrate on comprehensive features during the feature extraction, crucial for fault analysis. A multi-head self-attention mechanism regulated by a sliding window is utilized to expand the observation window's scope. Moreover, an asymptotic feature pyramid network is introduced to augment spatial feature extraction for smaller targets. Within this network architecture, adjacent low-level features are merged, while high-level features are gradually integrated into the fusion process. This prevents loss or degradation of feature information during transmission and interaction, enabling accurate localization of smaller targets. Drawing from wheel-rail faults of lifting equipment as an illustration, the proposed method is employed to diagnose an expanded fault dataset generated through transfer learning. Experimental findings substantiate that the proposed method in adeptly addressing numerous challenges encountered in the intelligent fault detection of special equipment. Moreover, it outperforms mainstream target detection models, achieving real-time detection capabilities.

BrABF3 promotes flowering through the direct activation of CONSTANS transcription in pak choi.

Drought stress triggers the accumulation of the phytohormone abscisic acid (ABA), which in turn activates the expression of the floral integrator gene CONSTANS (CO), accelerating flowering. However, the molecular mechanism of ABA-induced CO activation remains elusive. Here, we conducted a yeast one-hybrid assay using the CO promoter from Brassica campestris (syn. Brassica rapa) ssp. chinensis (pak choi) to screen the ABA-induced pak choi library and identified the transcription activator ABF3 (BrABF3). BrABF3, the expression of which was induced by ABA in pak choi, directly bound to the CO promoter from both pak choi and Arabidopsis. The BrABF3 promoter is specifically active in the Arabidopsis leaf vascular tissue, where CO is mainly expressed. Impaired BrABF3 expression in pak choi decreased BrCO expression levels and delayed flowering, whereas ectopic expression of BrABF3 in Arabidopsis increased CO expression and induced earlier flowering under the long-day conditions. Electrophoretic mobility shift assay analysis showed that BrABF3 was enriched at the canonical ABA-responsive element-ABRE binding factor (ABRE-ABF) binding motifs of the BrCO promoter. The direct binding of BrABF3 to the ABRE elements of CO was further confirmed by chromatin immunoprecipitation quantitative PCR. In addition, the induction of BrCO transcription by BrABF3 could be repressed by BrCDF1 in the morning. Thus, our results suggest that ABA could accelerate the floral transition by directly activating BrCO transcription through BrABF3 in pak choi.

Differential SW16.1 allelic effects and genetic backgrounds contributed to increased seed weight after soybean domestication.

Although seed weight has increased following domestication from wild soybean (Glycine soja) to cultivated soybean (Glycine max), the genetic basis underlying this change is unclear. Using mapping populations derived from chromosome segment substitution lines of wild soybean, we identified SW16.1 as the causative gene underlying a major quantitative trait locus controlling seed weight. SW16.1 encodes a nucleus-localized LIM domain-containing protein. Importantly, the GsSW16.1 allele from wild soybean accession N24852 had a negative effect on seed weight, whereas the GmSW16.1 allele from cultivar NN1138-2 had a positive effect. Gene expression network analysis, reverse-transcription quantitative polymerase chain reaction, and promoter-luciferase reporter transient expression assays suggested that SW16.1 regulates the transcription of MT4, a positive regulator of seed weight. The natural variations in SW16.1 and other known seed weight genes were analyzed in soybean germplasm. The SW16.1 polymorphism was associated with seed weight in 247 soybean accessions, showing much higher frequency of positive-effect alleles in cultivated soybean than in wild soybean. Interestingly, gene allele matrix analysis of the known seed weight genes revealed that G. max has lost 38.5% of the G. soja alleles and that most of the lost alleles had negative effects on seed weight. Our results suggest that eliminating negative alleles from G. soja led to a higher frequency of positive alleles and changed genetic backgrounds in G. max, which contributed to larger seeds in cultivated soybean after domestication from wild soybean. Our findings provide new insights regarding soybean domestication and should assist current soybean breeding programs.

Identification of Novel Genomic Regions for Bacterial Leaf Pustule (BLP) Resistance in Soybean (Glycine max L.) via Integrating Linkage Mapping and Association Analysis.

Bacterial leaf pustule (BLP), caused by Xanthornonas axonopodis pv. glycines (Xag), is a worldwide disease of soybean, particularly in warm and humid regions. To date, little is known about the underlying molecular mechanisms of BLP resistance. The only single recessive resistance gene rxp has not been functionally identified yet, even though the genotypes carrying the gene have been widely used for BLP resistance breeding. Using a linkage mapping in a recombinant inbred line (RIL) population against the Xag strain Chinese C5, we identified that quantitative trait locus (QTL) qrxp-17-2 accounted for 74.33% of the total phenotypic variations. We also identified two minor QTLs, qrxp-05-1 and qrxp-17-1, that accounted for 7.26% and 22.26% of the total phenotypic variations, respectively, for the first time. Using a genome-wide association study (GWAS) in 476 cultivars of a soybean breeding germplasm population, we identified a total of 38 quantitative trait nucleotides (QTNs) on chromosomes (Chr) 5, 7, 8, 9,15, 17, 19, and 20 under artificial infection with C5, and 34 QTNs on Chr 4, 5, 6, 9, 13, 16, 17, 18, and 20 under natural morbidity condition. Taken together, three QTLs and 11 stable QTNs were detected in both linkage mapping and GWAS analysis, and located in three genomic regions with the major genomic region containing qrxp_17_2. Real-time RT-PCR analysis of the relative expression levels of five potential candidate genes in the resistant soybean cultivar W82 following Xag treatment showed that of Glyma.17G086300, which is located in qrxp-17-2, significantly increased in W82 at 24 and 72 h post-inoculation (hpi) when compared to that in the susceptible cultivar Jack. These results indicate that Glyma.17G086300 is a potential candidate gene for rxp and the QTLs and QTNs identified in this study will be useful for marker development for the breeding of Xag-resistant soybean cultivars.

Reproductive developmental transcriptome analysis of Tripidium ravennae (Poaceae).

BACKGROUND: Tripidium ravennae is a cold-hardy, diploid species in the sugarcane complex (Poaceae subtribe Saccharinae) with considerable potential as a genetic resource for developing improved bioenergy and ornamental grasses. An improved understanding of the genetic regulation of reproductive processes (e.g., floral induction, inflorescence development, and seed development) will enable future applications of precision breeding and gene editing of floral and seed development. In particular, the ability to silence reproductive processes would allow for developing seedless forms of valuable but potentially invasive plants. The objective of this research was to characterize the gene expression environment of reproductive development in T. ravennae. RESULTS: During the early phases of inflorescence development, multiple key canonical floral integrators and pathways were identified. Annotations of type II subfamily of MADS-box transcription factors, in particular, were over-represented in the GO enrichment analyses and tests for differential expression (FDR p-value < 0.05). The differential expression of floral integrators observed in the early phases of inflorescence development diminished prior to inflorescence determinacy regulation. Differential expression analysis did not identify many unique genes at mid-inflorescence development stages, though typical biological processes involved in plant growth and development expressed abundantly. The increase in inflorescence determinacy regulatory elements and putative homeotic floral development unigenes at mid-inflorescence development coincided with the expression of multiple meiosis annotations and multicellular organism developmental processes. Analysis of seed development identified multiple unigenes involved in oxidative-reductive processes. CONCLUSION: Reproduction in grasses is a dynamic system involving the sequential coordination of complex gene regulatory networks and developmental processes. This research identified differentially expressed transcripts associated with floral induction, inflorescence development, and seed development in T. ravennae. These results provide insights into the molecular regulation of reproductive development and provide a foundation for future investigations and analyses, including genome annotation, functional genomics characterization, gene family evolutionary studies, comparative genomics, and precision breeding.

Rational design and testing of abiotic stress-inducible synthetic promoters from poplar cis-regulatory elements.

Abiotic stress resistance traits may be especially crucial for sustainable production of bioenergy tree crops. Here, we show the performance of a set of rationally designed osmotic-related and salt stress-inducible synthetic promoters for use in hybrid poplar. De novo motif-detecting algorithms yielded 30 water-deficit (SD) and 34 salt stress (SS) candidate DNA motifs from relevant poplar transcriptomes. We selected three conserved water-deficit stress motifs (SD18, SD13 and SD9) found in 16 co-expressed gene promoters, and we discovered a well-conserved motif for salt response (SS16). We characterized several native poplar stress-inducible promoters to enable comparisons with our synthetic promoters. Fifteen synthetic promoters were designed using various SD and SS subdomains, in which heptameric repeats of five-to-eight subdomain bases were fused to a common core promoter downstream, which, in turn, drove a green fluorescent protein (GFP) gene for reporter assays. These 15 synthetic promoters were screened by transient expression assays in poplar leaf mesophyll protoplasts and agroinfiltrated Nicotiana benthamiana leaves under osmotic stress conditions. Twelve synthetic promoters were induced in transient expression assays with a GFP readout. Of these, five promoters (SD18-1, SD9-2, SS16-1, SS16-2 and SS16-3) endowed higher inducibility under osmotic stress conditions than native promoters. These five synthetic promoters were stably transformed into Arabidopsis thaliana to study inducibility in whole plants. Herein, SD18-1 and SD9-2 were induced by water-deficit stress, whereas SS16-1, SS16-2 and SS16-3 were induced by salt stress. The synthetic biology design pipeline resulted in five synthetic promoters that outperformed endogenous promoters in transgenic plants.

Lipofection-mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells.

KEY MESSAGE: A novel and robust lipofection-mediated transfection approach for the use of DNA-free Cas9/gRNA RNP for gene editing has demonstrated efficacy in plant cells. Precise genome editing has been revolutionized by CRISPR/Cas9 systems. DNA-based delivery of CRISPR/Cas9 is widely used in various plant species. However, protein-based delivery of the in vitro translated Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complex into plant cells is still in its infancy even though protein delivery has several advantages. These advantages include DNA-free delivery, gene-edited host plants that are not transgenic, ease of use, low cost, relative ease to be adapted to high-throughput systems, and low off-target cleavage rates. Here, we show a novel lipofection-mediated transfection approach for protein delivery of the preassembled Cas9/gRNA RNP into plant cells for genome editing. Two lipofection reagents, Lipofectamine 3000 and RNAiMAX, were adapted for successful delivery into plant cells of Cas9/gRNA RNP. A green fluorescent protein (GFP) reporter was fused in-frame with the C-terminus of the Cas9 protein and the fusion protein was successfully delivered into non-transgenic tobacco cv. 'Bright Yellow-2' (BY2) protoplasts. The optimal efficiencies for Lipofectamine 3000- and RNAiMAX-mediated protein delivery were 66% and 48%, respectively. Furthermore, we developed a biolistic method for protein delivery based on the known proteolistics technique. A transgenic tobacco BY2 line expressing an orange fluorescence protein reporter pporRFP was targeted for knockout. We found that the targeted mutagenesis frequency for our Lipofectamine 3000-mediated protein delivery was 6%. Our results showed that the newly developed lipofection-mediated transfection approach is robust for the use of the DNA-free Cas9/gRNA technology for genome editing in plant cells.

Advanced genetic tools for plant biotechnology.

Basic research has provided a much better understanding of the genetic networks and regulatory hierarchies in plants. To meet the challenges of agriculture, we must be able to rapidly translate this knowledge into generating improved plants. Therefore, in this Review, we discuss advanced tools that are currently available for use in plant biotechnology to produce new products in plants and to generate plants with new functions. These tools include synthetic promoters, 'tunable' transcription factors, genome-editing tools and site-specific recombinases. We also review some tools with the potential to enable crop improvement, such as methods for the assembly and synthesis of large DNA molecules, plant transformation with linked multigenes and plant artificial chromosomes. These genetic technologies should be integrated to realize their potential for applications to pressing agricultural and environmental problems.

A profilin gene promoter from switchgrass (Panicum virgatum L.) directs strong and specific transgene expression to vascular bundles in rice.

KEY MESSAGE: A switchgrass vascular tissue-specific promoter (PvPfn2) and its 5'-end serial deletions drive high levels of vascular bundle transgene expression in transgenic rice. Constitutive promoters are widely used for crop genetic engineering, which can result in multiple off-target effects, including suboptimal growth and epigenetic gene silencing. These problems can be potentially avoided using tissue-specific promoters for targeted transgene expression. One particularly urgent need for targeted cell wall modification in bioenergy crops, such as switchgrass (Panicum virgatum L.), is the development of vasculature-active promoters to express cell wall-affective genes only in the specific tissues, i.e., xylem and phloem. From a switchgrass expression atlas we identified promoter sequence upstream of a vasculature-specific switchgrass profilin gene (PvPfn2), especially in roots, nodes and inflorescences. When the putative full-length (1715 bp) and 5'-end serial deletions of the PvPfn2 promoter (shortest was 413 bp) were used to drive the GUS reporter expression in stably transformed rice (Oryza sativa L.), strong vasculature-specificity was observed in various tissues including leaves, leaf sheaths, stems, and flowers. The promoters were active in both phloem and xylem. It is interesting to note that the promoter was active in many more tissues in the heterologous rice system than in switchgrass. Surprisingly, all four 5'-end promoter deletions, including the shortest fragment, had the same expression patterns as the full-length promoter and with no attenuation in GUS expression in rice. These results indicated that the PvPfn2 promoter variants are new tools to direct transgene expression specifically to vascular tissues in monocots. Of special interest is the very compact version of the promoter, which could be of use for vasculature-specific genetic engineering in monocots.

Synthetic TAL effectors for targeted enhancement of transgene expression in plants.

Transcription activator-like effectors (TALEs), secreted by the pathogenic bacteria Xanthomonas, specifically activate expression of targeted genes in plants. Here, we designed synthetic TALEs that bind to the flanking regions of the TATA-box motif on the CaMV 35S promoter for the purpose of understanding the engineerable 'hot-spots' for increasing transgene expression. We demonstrated that transient expression of de novo-engineered TALEs using agroinfiltration could significantly increase reporter gene expression in stable transgenic tobacco expressing the orange fluorescent protein reporter gene pporRFP under the control of synthetic inducible, minimal or full-length 35S promoters. Moreover, the additive effects of a combination of two different synthetic TALEs could significantly enhance the activation effects of TALEs on reporter gene expression more than when each TALE was used individually. We also studied the effects of the C-terminal domain and the activation domain of synthetic TALEs, as well as the best 'hot-spots' on the 35S promoter on targeted transgene activation. Furthermore, TALE activation of the Arabidopsis MYB transcription factor AtPAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT 1) in stable transgenic tobacco gave rise to a dark purple colour on infiltrated leaves when driven by four copies of cis-regulatory elements of pathogenesis-related gene (PR1) with enhancer motifs B and A1 from the 35S promoter. These results provide novel insights into the potential applications of synthetic TALEs for targeted gene activation of transgenes in plants.

The performance of pathogenic bacterial phytosensing transgenic tobacco in the field.

Phytosensors are useful for rapid-on-the-plant detection of contaminants and agents that cause plant stress. Previously, we produced a series of plant pathogen-inducible synthetic promoters fused to an orange fluorescent protein (OFP) reporter gene and transformed them into tobacco and Arabidopsis thaliana plants; in these transgenic lines, an OFP signal is expressed commensurate with the presence of plant pathogens. We report here the results of 2 years of field experiments using a subset of these bacterial phytosensing tobacco plants. Time-course analysis of field-grown phytosensors showed that a subset of plants responded predictably to treatments with Pseudomonas phytopathogens. There was a twofold induction in the OFP fluorescence driven by two distinct salicylic acid-responsive synthetic promoters, 4 × PR1 and 4 × SARE. Most notably, transgenic plants containing 4 × PR1 displayed the earliest and highest OFP induction at 48 and 72 h postinoculation (h p.i.) upon inoculation with two phytopathogens Pseudomonas syringae pv. tomato and P. syringae pv. tabaci, respectively. These results demonstrate transgenic tobacco harbouring a synthetic inducible promoter-driven OFP could be used to facilitate monitoring and early-warning reporting of phytopathogen infections in agricultural fields.

Computational discovery of soybean promoter cis-regulatory elements for the construction of soybean cyst nematode-inducible synthetic promoters.

Computational methods offer great hope but limited accuracy in the prediction of functional cis-regulatory elements; improvements are needed to enable synthetic promoter design. We applied an ensemble strategy for de novo soybean cyst nematode (SCN)-inducible motif discovery among promoters of 18 co-expressed soybean genes that were selected from six reported microarray studies involving a compatible soybean-SCN interaction. A total of 116 overlapping motif regions (OMRs) were discovered bioinformatically that were identified by at least four out of seven bioinformatic tools. Using synthetic promoters, the inducibility of each OMR or motif itself was evaluated by co-localization of gain of function of an orange fluorescent protein reporter and the presence of SCN in transgenic soybean hairy roots. Among 16 OMRs detected from two experimentally confirmed SCN-inducible promoters, 11 OMRs (i.e. 68.75%) were experimentally confirmed to be SCN-inducible, leading to the discovery of 23 core motifs of 5- to 7-bp length, of which 14 are novel in plants. We found that a combination of the three best tools (i.e. SCOPE, W-AlignACE and Weeder) could detect all 23 core motifs. Thus, this strategy is a high-throughput approach for de novo motif discovery in soybean and offers great potential for novel motif discovery and synthetic promoter engineering for any plant and trait in crop biotechnology.

Construction of a mitophagy-related prognostic signature for predicting prognosis and tumor microenvironment in lung adenocarcinoma.

Background: Mitophagy is the selective degradation of mitochondria by autophagy. It becomes increasingly clear that mitophagy pathways are important for cancer cells to adapt to their high-energy needs. However, which genes associated with mitophagy could be used to prognosis cancer is unknown. Methods: We created a clinical prognostic model using mitophagy-related genes (MRGs) in lung adenocarcinoma (LUAD) patients for the first time, and we employed bioinformatics methods to search for biomarkers that affect the progression and prognosis of LUAD. Transcriptome data for LUAD were obtained from The Cancer Genome Atlas (TCGA) database, and additional expression data from LUAD patients were sourced from the Gene Expression Omnibus (GEO) database. Furthermore, 25 complete MRGs were identified based on annotations from the MSigDB database. Results: A comparison of the mitophagy scores between the groups with high and low scores was done using receiver operating characteristic (ROC) curves, which also revealed the differential gene expression patterns between the two groups. Using Kaplan-Meier analysis, two prognostic MRGs from the groups with high and low mitophagy scores were identified: TOMM40 and VDAC1. Using univariate and multivariate Cox regression, the relationship between the expression levels of these two genes and prognostic clinical features of LUAD was examined further.The prognosis of LUAD patients was shown to be significantly correlated (P < 0.05) with the expression levels of these two genes. Conclusions: Our prognostic model would improve the prognosis of LUAD and guide clinical treatments.

Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode.

Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in soybean defence against soybean cyst nematode (Heterodera glycines Ichinohe, SCN). GmSAMT1 was identified as a candidate SCN defence-related gene in our previous analysis of soybean defence against SCN using GeneChip microarray experiments. The current study started with the isolation of the full-length cDNAs of GmSAMT1 from a SCN-resistant soybean line and from a SCN-susceptible soybean line. The two cDNAs encode proteins of identical sequences. The GmSAMT1 cDNA was expressed in Escherichia coli. Using in vitro enzyme assays, E. coli-expressed GmSAMT1 was confirmed to function as salicylic acid methyltransferase. The apparent Km value of GmSAMT1 for salicylic acid was approximately 46 µM. To determine the role of GmSAMT1 in soybean defence against SCN, transgenic hairy roots overexpressing GmSAMT1 were produced and tested for SCN resistance. Overexpression of GmSAMT1 in SCN-susceptible backgrounds significantly reduced the development of SCN, indicating that overexpression of GmSAMT1 in the transgenic hairy root system could confer resistance to SCN. Overexpression of GmSAMT1 in transgenic hairy roots was also found to affect the expression of selected genes involved in salicylic acid biosynthesis and salicylic acid signal transduction.

Bacterial pathogen phytosensing in transgenic tobacco and Arabidopsis plants.

Plants are subject to attack by a wide range of phytopathogens. Current pathogen detection methods and technologies are largely constrained to those occurring post-symptomatically. Recent efforts were made to generate plant sentinels (phytosensors) that can be used for sensing and reporting pathogen contamination in crops. Engineered phytosensors indicating the presence of plant pathogens as early-warning sentinels potentially have tremendous utility as wide-area detectors. We previously showed that synthetic promoters containing pathogen and/or defence signalling inducible cis-acting regulatory elements (RE) fused to a fluorescent protein (FP) reporter could detect phytopathogenic bacteria in a transient phytosensing system. Here, we further advanced this phytosensing system by developing stable transgenic tobacco and Arabidopsis plants containing candidate constructs. The inducibility of each synthetic promoter was examined in response to biotic (bacterial pathogens) or chemical (plant signal molecules salicylic acid, ethylene and methyl jasmonate) treatments using stably transgenic plants. The treated plants were visualized using epifluorescence microscopy and quantified using spectrofluorometry for FP synthesis upon induction. Time-course analyses of FP synthesis showed that both transgenic tobacco and Arabidopsis plants were capable to respond in predictable ways to pathogen and chemical treatments. These results provide insights into the potential applications of transgenic plants as phytosensors and the implementation of emerging technologies for monitoring plant disease outbreaks in agricultural fields.

The presence of multiple introns is essential for ERECTA expression in Arabidopsis.

Gene expression in eukaryotes is often enhanced by the presence of introns. Depending on the specific gene, this enhancement can be minor or very large and occurs at both the transcriptional and post-transcriptional levels. The Arabidopsis ERECTA gene contains 27 exons encoding a receptor-like kinase that promotes cell proliferation and inhibits cell differentiation in above-ground plant organs. The expression of ERECTA very strongly depends on the presence of introns. The intronless ERECTA gene does not rescue the phenotype of erecta mutant plants and produces about 500-900 times less protein compared with the identical construct containing introns. This result is somewhat surprising as the region upstream of the ERECTA coding sequence effectively promotes the expression of extraneous genes. Here, we demonstrate that introns are essential for ERECTA mRNA accumulation and, to a lesser extent, for mRNA utilization in translation. Since mRNA produced by intronless ERECTA is degraded at the 3' end, we speculate that introns increase mRNA accumulation through increasing its stability at least in part. No individual intron is absolutely necessary for ERECTA expression, but rather multiple introns in specific locations increase ERECTA expression in an additive manner. The ability of introns to promote ERECTA expression might be linked to the process of splicing and not to a particular intron sequence.

Stepwise Optimization of Real-Time RT-PCR Analysis.

Quantitative real-time reverse transcription PCR (qRT-PCR) analysis has been used routinely to quantify gene expression levels. Primer design and the optimization of qRT-PCR parameters are critical for the accuracy and reproducibility of qRT-PCR analysis. Computational tool-assisted primer design often overlooks the presence of homologous sequences of the gene of interest and the sequence similarities between homologous genes in a plant genome. This sometimes results in skipping the optimization of qRT-PCR parameters due to the false confidence in the quality of the designed primers. Here we present a stepwise optimization protocol for single nucleotide polymorphisms (SNPs)-based sequence-specific primer design and sequential optimization of primer sequences, annealing temperatures, primer concentrations, and cDNA concentration range for each reference and target gene. The goal of this optimization protocol is to achieve a standard cDNA concentration curve with an R2 ≥ 0.9999 and efficiency (E) = 100 ± 5% for the best primer pair of each gene, which serves as the prerequisite for using the 2-ΔΔCT method for data analysis.

Engineered Cleistogamy in Camelina sativa for bioconfinement.

Camelina sativa is a self-pollinating and facultative outcrossing oilseed crop. Genetic engineering has been used to improve camelina yield potential for altered fatty acid composition, modified protein profiles, improved seed and oil yield, and enhanced drought resistance. The deployment of transgenic camelina in the field posits high risks related to the introgression of transgenes into non-transgenic camelina and wild relatives. Thus, effective bioconfinement strategies need to be developed to prevent pollen-mediated gene flow (PMGF) from transgenic camelina. In the present study, we overexpressed the cleistogamy (i.e. floral petal non-openness)-inducing PpJAZ1 gene from peach in transgenic camelina. Transgenic camelina overexpressing PpJAZ1 showed three levels of cleistogamy, affected pollen germination rates after anthesis but not during anthesis, and caused a minor silicle abortion only on the main branches. We also conducted field trials to examine the effects of the overexpressed PpJAZ1 on PMGF in the field, and found that the overexpressed PpJAZ1 dramatically inhibited PMGF from transgenic camelina to non-transgenic camelina under the field conditions. Thus, the engineered cleistogamy using the overexpressed PpJAZ1 is a highly effective bioconfinement strategy to limit PMGF from transgenic camelina, and could be used for bioconfinement in other dicot species.