Comparative transcriptome analysis and genetic dissection of vegetative branching traits in foxtail millet (Setaria italica).

KEY MESSAGE: Two major genetic loci, qTN5.1 and qAB9.1, were identified and finely mapped to the 255 Kb region with one potential candidate gene for tiller number and the 521 Kb region with eight candidate genes for axillary branch number, respectively. Vegetative branching including tillering and axillary branching are vital traits affecting both the plant architecture and the biomass in cereal crops. However, the mechanism underlying the formation of vegetative branching in foxtail millet is largely unknown. Here, a foxtail millet cultivar and its bushy wild relative Setaria viridis accession were used to construct segregating populations to identify candidate genes regulating tiller number and axillary branch number. Transcriptome analysis using vegetative branching bud samples of parental accessions was performed, and key differentially expressed genes and pathways regulating vegetative branching were pointed out. Bulk segregant analysis on their F2:3 segregating population was carried out, and a major QTL for tiller number (qTN5.1) and two major QTLs for axillary branch number (qAB2.1 and qAB9.1) were detected. Fine-mapping strategy was further performed on F2:4 segregate population, and Seita.5G356600 encoding ß-glucosidase 11 was identified as the promising candidate gene for qTN5.1, and eight genes, especially Seita.9G125300 and Seita.9G125400 annotated as B-S glucosidase 44, were finally identified as candidate genes for regulating axillary branching. Findings in this study will help to elucidate the genetic basis of the vegetative branching formation of foxtail millet and lay a foundation for breeding foxtail millet varieties with ideal vegetative branching numbers.

Genome-wide identification of GRF transcription factors and their expression profile in stem meristem of broomcorn millet (Panicum miliaceum L.).

To understand the genome-wide information of the GRF family genes in broomcorn millet and their expression profile in the vegetative meristems, bioinformatic methods and transcriptome sequencing were used to analyze the characteristics, physical and chemical properties, phylogenetic relationship, chromosome distribution, gene structure, cis-acting elements and expression profile in stem meristem for the GRF family members. The results showed that the GRF gene family of millet contains 21 members, and the PmGRF gene is unevenly distributed on 12 chromosomes. The lengths of PmGRF proteins vary from 224 to 618 amino acids, and the isoelectric points are between 4.93-9.69. Each member of the family has 1-4 introns and 2-5 exons. The protein PmGRF13 is localized in both the nucleus and chloroplast, and the rest PmGRF proteins are located in the nucleus. Phylogenetic analysis showed that the 21 GRF genes were divided into 4 subfamilies (A,B,C and D) in broomcorn millet. The analysis of cis-acting elements showed that there were many cis-acting elements involved in light response, hormone response, drought induction, low temperature response and other environmental stress responses in the 2000 bp sequence upstream of the GRF genes. Transcriptome sequencing and qRT-PCR analyses showed that the expression levels of PmGRF3 and PmGRF12 in the dwarf variety Zhang778 were significantly higher than those of the tall variety Longmi12 in the internode and node meristems at the jointing stage, while the expression patterns of PmGRF4, PmGRF16 and PmGRF21 were reverse. In addition, the expression levels of PmGRF2 and PmGRF5 in the internode of Zhang778 were significantly higher than Longmi12. The other GRF genes were not or insignificantly expressed. These results indicated that seven genes, PmGRF2, PmGRF3, PmGRF4, PmGRF5, PmGRF12, PmGRF16 and PmGRF21, were related to the formation of plant height in broomcorn millet.

Genome-wide identification of quantitative trait loci for morpho-agronomic and yield-related traits in foxtail millet (Setaria italica) across multi-environments.

Foxtail millet (Setaria italica) is an ideal model of genetic system for functional genomics of the Panicoideae crop. Identification of QTL responsible for morpho-agronomic and yield-related traits facilitates dissection of genetic control and breeding in cereal crops. Here, based on a Yugu1 × Longgu7 RIL population and genome-wide resequencing data, an updated linkage map harboring 2297 bin and 74 SSR markers was constructed, spanning 1315.1 cM with an average distance of 0.56 cM between adjacent markers. A total of 221 QTL for 17 morpho-agronomic and yield-related traits explaining 5.5 ~ 36% of phenotypic variation were identified across multi-environments. Of these, 109 QTL were detected in two to nine environments, including the most stable qLMS6.1 harboring a promising candidate gene Seita.6G250500, of which 70 were repeatedly identified in different trials in the same geographic location, suggesting that foxtail millet has more identical genetic modules under the similar ecological environment. One hundred-thirty QTL with overlapping intervals formed 22 QTL clusters. Furthermore, six superior recombinant inbred lines, RIL35, RIL48, RIL77, RIL80, RIL115 and RIL125 with transgressive inheritance and enrichment of favorable alleles in plant height, tiller, panicle morphology and yield related-traits were screened by hierarchical cluster. These identified QTL, QTL clusters and superior lines lay ground for further gene-trait association studies and breeding practice in foxtail millet.

Overexpression of miR-138-5p Sensitizes Taxol-Resistant Epithelial Ovarian Cancer Cells through Targeting Cyclin-Dependent Kinase 6.

BACKGROUND: Ovarian cancer, one of the most malignant diseases in female, is associated with poor diagnosis and low 5-year survival rate. Taxol is a widely used chemotherapeutic drug for the treatment of ovarian cancer by targeting the microtubules of the mitotic spindle to induce cancer cell death. However, with the widespread clinical applications of Taxol, a large fraction of ovarian cancer patients developed drug resistance. RESULTS: Here, we report miR-138-5p is significantly downregulated in epithelial ovarian cancer tissues compared with their matched normal ovarian tissues. Overexpression of miR-138-5p effectively sensitized ovarian cancer cells to Taxol. By establishing Taxol-resistant cell line from the epithelial ovarian cancer cell line, HO-8910, we found miR-138-5p was significantly downregulated in Taxol-resistant cells. Furthermore, overexpression of miR-138-5p dramatically overcame the chemoresistance of Taxol-resistant cells. Intriguingly, bioinformatic analysis indicated miR-138-5p had putative binding sites for cyclin-dependent kinase 6 (CDK6). This negative regulation was further verified from epithelial ovarian cancer tissues. Luciferase assay demonstrated miR-138-5p could directly bind to 3'UTR of CDK6. Importantly, silencing CDK6 expression by siRNA successfully increased the sensitivity of both parental and Taxol-resistant ovarian cancer cells. Finally, rescue experiments clearly elucidated restoration of CDK6 in miR-138-5p-overexpressing ovarian cancer cells successfully recovered the Taxol resistance. CONCLUSION: In summary, these findings suggest important molecular mechanisms for the miR-138-5p-mediated Taxol sensitivity of ovarian cancer via directly targeting CDK6, suggesting miR-138-5p is an effective therapeutic target for the noncoding RNA-based anti-chemoresistance treatment.

QTL mapping of yield component traits on bin map generated from resequencing a RIL population of foxtail millet (Setaria italica).

BACKGROUND: Foxtail millet (Setaria italica) has been developed into a model genetical system for deciphering architectural evolution, C4 photosynthesis, nutritional properties, abiotic tolerance and bioenergy in cereal grasses because of its advantageous characters with the small genome size, self-fertilization, short growing cycle, small growth stature, efficient genetic transformation and abundant diverse germplasm resources. Therefore, excavating QTLs of yield component traits, which are closely related to aspects mentioned above, will further facilitate genetic research in foxtail millet and close cereal species. RESULTS: Here, 164 Recombinant inbreed lines from a cross between Longgu7 and Yugu1 were created and 1,047,978 SNPs were identified between both parents via resequencing. A total of 3413 bin markers developed from SNPs were used to construct a binary map, containing 3963 recombinant breakpoints and totaling 1222.26 cM with an average distance of 0.36 cM between adjacent markers. Forty-seven QTLs were identified for four traits of straw weight, panicle weight, grain weight per plant and 1000-grain weight. These QTLs explained 5.5-14.7% of phenotypic variance. Thirty-nine favorable QTL alleles were found to inherit from Yugu1. Three stable QTLs were detected in multi-environments, and nine QTL clusters were identified on Chromosome 3, 6, 7 and 9. CONCLUSIONS: A high-density genetic map with 3413 bin markers was constructed and three stable QTLs and 9 QTL clusters for yield component traits were identified. The results laid a powerful foundation for fine mapping, identifying candidate genes, elaborating molecular mechanisms and application in foxtail millet breeding programs by marker-assisted selection.

A high density genetic map and QTL for agronomic and yield traits in Foxtail millet [Setaria italica (L.) P. Beauv].

BACKGROUND: Foxtail millet [Setaria italica (L.) P. Beauv.], a crop of historical importance in China, has been adopted as a model crop for studying C-4 photosynthesis, stress biology and biofuel traits. Construction of a high density genetic map and identification of stable quantitative trait loci (QTL) lay the foundation for marker-assisted selection for agronomic traits and yield improvement. RESULT: A total of 10598 SSR markers were developed according to the reference genome sequence of foxtail millet cultivar 'Yugu1'. A total of 1013 SSR markers showing polymorphism between Yugu1 and Longgu7 were used to genotype 167 individuals from a Yugu1 × Longgu7 F2 population, and a high density genetic map was constructed. The genetic map contained 1035 loci and spanned 1318.8 cM with an average distance of 1.27 cM between adjacent markers. Based on agronomic and yield traits identified in 2 years, 29 QTL were identified for 11 traits with combined analysis and single environment analysis. These QTL explained from 7.0 to 14.3 % of phenotypic variation. Favorable QTL alleles for peduncle length originated from Longgu7 whereas favorable alleles for the other traits originated from Yugu1 except for qLMS6.1. CONCLUSIONS: New SSR markers, a high density genetic map and QTL identified for agronomic and yield traits lay the ground work for functional gene mapping, map-based cloning and marker-assisted selection in foxtail millet.

Genetic Diversity and Population Structure of Broomcorn Millet (Panicum miliaceum L.) Cultivars and Landraces in China Based on Microsatellite Markers.

Broomcorn millet (Panicum miliaceum L.), one of the first domesticated crops, has been grown in Northern China for at least 10,000 years. The species is presently a minor crop, and evaluation of its genetic diversity has been very limited. In this study, we analyzed the genetic diversity of 88 accessions of broomcorn millet collected from various provinces of China. Amplification with 67 simple sequence repeat (SSR) primers revealed moderate levels of diversity in the investigated accessions. A total of 179 alleles were detected, with an average of 2.7 alleles per locus. Polymorphism information content and expected heterozygosity ranged from 0.043 to 0.729 (mean = 0.376) and 0.045 to 0.771 (mean = 0.445), respectively. Cluster analysis based on the unweighted pair group method of mathematical averages separated the 88 accessions into four groups at a genetic similarity level of 0.633. A genetic structure assay indicated a close correlation between geographical regions and genetic diversity. The uncovered information will be valuable for defining gene pools and developing breeding programs for broomcorn millet. Furthermore, the millet-specific SSR markers developed in this study should serve as useful tools for assessment of genetic diversity and elucidation of population structure in broomcorn millet.

Clinical scoring model based on age, NIHSS, and stroke-history predicts outcome 3 months after acute ischemic stroke.

Background: The clinical nomogram is a popular decision-making tool that can be used to predict patient outcomes, bringing benefits to clinicians and patients in clinical decision-making. This study established a simple and effective clinical prediction model to predict the 3-month prognosis of acute ischemic stroke (AIS), and based on the predicted results, improved clinical decision-making and improved patient outcomes. Methods: From 18 December 2021 to 8 January 2022, a total of 146 hospitalized patients with AIS confirmed by brain MR were collected, of which 132 eligible participants constituted a prospective study cohort. The least absolute shrinkage and selection operator (LASSO) regression was applied to a nomogram model development dataset to select features associated with poor prognosis in AIS for inclusion in the logistic regression of our risk scoring system. On this basis, the nomogram was drawn, evaluated for discriminative power, calibration, and clinical benefit, and validated internally by bootstrap. Finally, the optimal cutoff point for each independent risk factor and nomogram was calculated using the Youden index. Results: A total of 132 patients were included in this study, including 85 men and 47 women. Good outcome was found in 94 (71.212%) patients and bad outcome in 38 (28.788%) patients during the follow-up period. A total of eight (6.061%) deaths were reported over this period, of whom five (3.788%) died during hospitalization. Five factors affecting the 3-month prognosis of AIS were screened by LASSO regression, namely, age, hospital stay, previous stroke, atrial fibrillation, and NIHSS. Further multivariate logistic regression revealed three independent risk factors affecting patient outcomes, namely, age, previous stroke, and NIHSS. The area under the curve of the nomogram was 0.880, and the 95% confidence interval was 0.818-0.943, suggesting that the nomogram model has good discriminative power. The p-value for the calibration curve is 0.925, indicating that the nomogram model is well-calibrated. According to the decision curve analysis results, when the threshold probability is >0.01, the net benefit obtained by the nomogram is the largest. The concordance index for 1,000 bootstrapping calculations is 0.869. The age cutoff for predicting poor patient outcomes using the Youden index was 76.5 years (specificity 0.777 and sensitivity 0.684), the cutoff for the NIHSS was 7.5 (specificity 0.936, sensitivity 0.421), and the cutoff for total nomogram score was 68.8 (sensitivity 81.6% and specificity 79.8%). Conclusion: The nomogram model established in this study had good discrimination, calibration, and clinical benefits. A nomogram composed of age, previous stroke, and NIHSS might predict the prognosis of stroke after AIS. It might intuitively and individually predict the risk of poor prognosis in 3 months of AIS and provide a reference basis for screening the treatment plan of patients.

Identification and fine-mapping of a major QTL (PH1.1) conferring plant height in broomcorn millet (Panicum miliaceum).

The plant height of broomcorn millet (Panicum miliaceum) is a significant agronomic trait that is closely related to its plant architecture, lodging resistance, and final yield. However, the genes underlying the regulation of plant height in broomcorn millet are rarely reported. Here, an F2 population derived from a cross between a normal variety, "Longmi12," and a dwarf mutant, "Zhang778," was constructed. Genetic analysis for the F2 and F2:3 populations revealed that the plant height was controlled by more than one locus. A major quantitative trait locus (QTL), PH1.1, was preliminarily identified in chromosome 1 using bulked segregant analysis sequencing (BSA-seq). PH1.1 was fine-mapped to a 109-kb genomic region with 15 genes using a high-density map. Among them, longmi011482 and longmi011489, containing nonsynonymous variations in their coding regions, and longmi011496, covering multiple insertion/deletion sequences in the promoter regions, may be possible candidate genes for PH1.1. Three diagnostic markers closely linked to PH1.1 were developed to validate the PH1.1 region in broomcorn millet germplasm. These findings laid the foundation for further understanding of the molecular mechanism of plant height regulation in broomcorn millet and are also beneficial to the breeding program for developing new varieties with optimal height.

Association of HLA-DM and HLA class II genes with antibody response induced by inactivated Japanese encephalitis vaccine.

HLA class II molecules, HLA-DR, DP and DQ, together with HLA II-like protein DM, play a dominant role in the processing and presentation of antigens, which may influence vaccine effectiveness. We previously demonstrated that variations in the HLA-DRB1, DPB1 and DQB1 genes may affect the neutralising antibody (NAb) response induced by the inactivated Japanese encephalitis vaccine (IJEV). In the present study, we genotyped HLA-DPA1, DQA1, DMA and DMB genes and used previous HLA-DRB1, DPB1 and DQB1 data to evaluate the association of these genes with IJEV-induced NAbs, at both the seroconversion and geometric mean titres (GMTs). We confirmed the seropositive association of DQB1*02:01 and NAbs (0.156 vs. 0.075, p_adj = 0.018; OR = 2.270; 95% CI = 1.285-3.999) and seronegative association of DQB1*02:02 (0.014 vs. 0.09, p_adj = 0.0002; OR = 0.130; 95% CI = 0.047-0.400). Furthermore, the DMB*01:03-DMA*01:01-DPA1*01:03-DPB1*04:01 haplotype was associated with a negative response (0.020 vs. 0.074; p_adj = 0.03; OR = 0.250; 95% CI = 0.097-0.649), whereas DRB1*15:02-DMB*01:01-DMA*01:01 was associated with a positive response (0.034 vs. 0; p_adj = 0.044). In addition, DRB1*12:02, DRB1*13:02, DPB1*04:01, DPB1*05:01, DPB1*09:01, DQA1*06:01 and DQA1*01:02 were associated with a higher GMT of NAbs, whereas DRB1*11:01, DPB1*13:01 and DQA1*05:05 were associated with a lower GMT of NAbs. In conclusion, the present study suggests that variations in the HLA-DM and HLA class II genes, as well as their combined allotypes, may influence the IJEV NAbs at seroconversion and GMT levels.

Minocycline attenuates neuronal apoptosis and improves motor function after traumatic brain injury in rats.

Minocycline is a type of tetracycline antibiotic with broad-spectrum antibacterial activity that has been demonstrated to protect the brain against a series of central nervous system diseases. However, the precise mechanisms of these neuroprotective actions remain unknown. In the present study, we found that minocycline treatment significantly reduced HT22 cell apoptosis in a mechanical cell injury model. In addition, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining confirmed the neuroprotective effects of minocycline in vivo through the inhibition of apoptosis in a rat model of controlled cortical impact (CCI) brain injury. The western blotting analysis revealed that minocycline treatment significantly downregulated the pro-apoptotic proteins BAX and cleaved caspase-3 and upregulated the anti-apoptotic protein BCL-2. Furthermore, the beam-walking test showed that the administration of minocycline ameliorated traumatic brain injury (TBI)-induced deficits in motor function. Taken together, these findings suggested that minocycline attenuated neuronal apoptosis and improved motor function following TBI.

Genetic Diversity and Classification of the Cytoplasm of Chinese Elite Foxtail Millet [Setaria italica (L.) P. Beauv.] Parental Lines Revealed by Chloroplast Deoxyribonucleic Acid Variation.

Due to the maternal inheritance of cytoplasm, using foxtail millet [Setaria italica (L.) P. Beauv.] male sterile lines with a single cytoplasmic source as the female parent will inevitably lead to a narrow source of cytoplasm in hybrids, which may make them vulnerable to infection by cytoplasm-specific pathogens, ultimately leading to destructive yield losses. To assess cytoplasmic genetic diversity in plants, molecular markers derived from chloroplast DNA (cpDNA) have been used. However, such markers have not yet been applied to foxtail millet. In this study, we designed and screened nine pairs of polymorphic foxtail millet-specific primers based on its completely sequenced cpDNA. Using these primers, we analyzed the genetic diversity and cytoplasmic types of 130 elite foxtail millet parental lines collected in China. Our results revealed that the cytoplasmic genetic diversity of these accessions was low and needs to be increased. The parental lines were divided into four cytoplasmic types according to population structure analysis and a female parent-derivative evolutionary graph, indicating that the cytoplasmic types of elite foxtail millet lines were rather limited. A principal component analysis (PCA) plot was linked with the geographic and ecological distribution of accessions for each cytoplasmic type, as well as their basal maternal parents. Collectively, our results suggest that enriching cytoplasmic sources through the use of accessions from diverse ecological regions and other countries as the female parent may improve foxtail millet breeding programs, and prevent infection by cytoplasm-specific pathogens.

Determination of multiresidue of avermectins in bovine liver by an indirect competitive ELISA.

An ELISA was developed to detect multiresidues of avermectins (AVMs) including abamectin (ABM), ivermectin (IVM), and eprinomectin (EPR) in bovine liver. The modified ABM, 4'-O-succinoyl-ABM was conjugated to bovine serum albumin as the immunogen for the preparation of polyclonal antibodies to AVMs and conjugated to ovalbumin as the coating antigen for the ELISA. Serum with the highest antibody titers to AVMs, which had a cross-reactivity of 100% with ABM, 145.4% with EPR, and 25% for IVM, was selected for the development of an indirect competitive ELISA. The ELISA could detect ABM, IVM, and EPR residues in bovine liver tissues, with a limit of quantitation of 1.06 ng/mL for all three AVMs. Optimal pH, ion strength, organic solvent, and duration of incubations were investigated to increase the sensitivity of the ELISA. Recoveries of these drugs ranged from 53.8% to 80.6% with inter-assay coefficients of variation (CV) of 3.4-17.9% and intra-assay CV of 5.5-14.7%. Analysis results of field samples by the ELISA were consistent with those by a previously developed HPLC method. The ELISA can be used as a rapid method for screening of AVMs residues in bovine liver.

Application of quantum dot-antibody conjugates for detection of sulfamethazine residue in chicken muscle tissue.

A new indirect competitive fluorescence-linked immunosorbent assay (cFLISA) method for the detection of sulfamethazine (SM2) in chicken muscle tissue was demonstrated using quantum dots (QDs) as the fluorescence label coupled with secondary antibody. The sensitivity of the cFLISA was compared with that of the HPLC method. The 50% inhibition value (IC50) and the limit of detection (LOD) of the cFLISA were 7.7 and 1.0 ng/mL, respectively. When SM2 was spiked at levels of 50, 100, and 200 ng/g, recoveries ranged from 80.6% to 117.4%, with a coefficient of variation of 6.9-9.6%. In the incurred sample analysis, the amounts of SM2 quantified by cFLISA were similar to the results obtained by the HPLC method. This study shows that cFLISA could be used as a screening method in practice.

Residue depletion of eprinomectin in bovine tissues after subcutaneous administration.

A study of the tissue depletion of eprinomectin (EPR) subcutaneously administered to cattle at a dose of 500 mg per kg of body weight was carried out. EPR concentrations were determined in muscle, liver, kidney, and fat. Twenty-four parasite-free cross cattle were treated with the EPR injectable oil formulation. Three treated animals (two males and one female) were selected randomly to be sacrificed at 1, 3, 7, 14, 21, 28, 42, and 56 days withdrawal after injection. EPR residue concentrations were determined using HPLC with fluorescence detection. Muscle samples showed the lowest EPR concentrations throughout the study period. The highest EPR concentrations at all sampling times were measured in liver tissue, indicating that liver is a target tissue for EPR. EPR concentrations in all of the tissues analyzed were below the accepted maximum residue limits recommended by the European Union at 8 days posttreatment.

Multiresidue analysis of avermectins in bovine liver by immunoaffinity column cleanup procedure and liquid chromatography with fluorescence detector.

A multiresidue liquid chromatography (LC) method was developed to detect avermectin, ivermectin, doramectin, and eprinomectin simultaneously in bovine liver. The extracted samples were cleaned up by an immunoaffinity column, which was prepared by coupling anti-avermectin polyclonal antibody with CNBr-activated Sepharose 4B. The dynamic column capacities of avermectin, ivermectin, doramectin, and eprinomectin were 3531, 3542, 3543, and 3284 nglmL gel, respectively. The eluate was evaporated to dryness, and residues were derivatized and determined by LC with fluorescence detector set at 365 nm excitation and 465 nm emission wavelengths. Recoveries ranged from 79.3 to 115.9% with coefficients of variation of 1.1-19.4% when avermectin, ivermectin, doramectin, and eprinomectin were spiked at levels of 5-100 ng/g. The limit of quantitation of the method was 2 ng/g for each drug.

[Immunogenicity of sabin inactivated poliovirus vaccine induced by diphtheria-tetanus-acellular pertussis and Sabin inactivated poliovirus combined vaccine].

OBJECTIVE: In order to search the preparation process and optimazing dosage ratio of adsorbed diphtheria-tetanus-acellular pertussis and sabin inactivated poliovirus combined vaccine (DTaP-sIPV), the neutralizing antibody titers of IPV induced by different concentration of DTaP-sIPV were investigated on rats. METHODS: Two batches of DTaP-sLPV were produced using different concentration of sIPV and the quality control was carried. Together with sabin-IPV and DTaP-wIPV ( boostrix-polio, GSK, Belgium) as control group, the DTaP-sIPV were administrated on three-dose schedule at 0, 1, 2 month on rats. Serum sample were collected 30 days after each dose and neutralizing antibody titers against three types poliovirus were determined using micro-neutralization test. RESULTS: Two batches of prepared DTaP-sIPV and control sLPV were according to the requirement of Chinese Pharmacopoeia (Volume III, 2005 edition) and showed good stability. The seropositivity rates were 100% for sabin inactivated poliovirus antigen in all groups. The GMTs (Geometric mean titers) of neutralizing antibodies against three types poliovirus increased. CONCLUSION: The prepared DTaP-sIPV was safe, stable and effective and could induced high level neutralizing antibody against poliovirus on rats.