Integrative analyses of long and short-read RNA sequencing reveal the spliced isoform regulatory network of seedling growth dynamics in upland cotton.

The polyploid genome of cotton has significantly increased the transcript complexity. Recent advances in full-length transcript sequencing are now widely used to characterize the complete landscape of transcriptional events. Such studies in cotton can help us to explore the genetic mechanisms of the cotton seedling growth. Through long-read single-molecule RNA sequencing, this study compared the transcriptomes of three yield contrasting genotypes of upland cotton. Our analysis identified different numbers of spliced isoforms from 31,166, 28,716, and 28,713 genes in SJ48, Z98, and DT8 cotton genotypes, respectively, most of which were novel compared to previous cotton reference transcriptomes, and showed significant differences in the number of exon structures and coding sequence length due to intron retention. Quantification of isoform expression revealed significant differences in expression in the root and leaf of each genotype. An array of key isoform target genes showed protein kinase or phosphorylation functions, and their protein interaction network contained most of the circadian oscillator proteins. Spliced isoforms from the GIGANTEA (GI) protien were differentially regulated in each genotype and might be expected to regulate translational activities, including the sequence and function of target proteins. In addition, these spliced isoforms generate diurnal expression profiles in cotton leaves, which may alter the transcriptional regulatory network of seedling growth. Silencing of the novel spliced GI isoform Gh_A02G0645_N17 significantly affected biomass traits, contributed to variable growth, and increased transcription of the early flowering pathway gene ELF in cotton. Our high-throughput hybrid sequencing results will be useful to dissect functional differences among spliced isoforms in the polyploid cotton genome.

Deciphering the dynamic expression network of fiber elongation and the functional role of the GhTUB5 gene for fiber length in cotton based on an introgression population of upland cotton.

INTRODUCTION: Interspecific introgression between Gossypium hirsutum and G. barbadense allows breeding cotton with outstanding fiber length (FL). However, the dynamic gene regulatory network of FL-related genes has not been characterized, and the functional mechanism through which the hub gene GhTUB5 mediates fiber elongation has yet to be determined. METHODS: Coexpression analyses of 277 developing fiber transcriptomes integrated with QTL mapping using 250 introgression lines of different FL phenotypes were conducted to identify genes related to fiber elongation. The function of GhTUB5 was determined by ectopic expression of two TUB5 alleles in Arabidopsis and knockout of GhTUB5 in upland cotton. Yeast two-hybrid, split-luciferase and pull-down assays were conducted to screen for interacting proteins, and upstream genes were identified by yeast one-hybrid, dual-LUC and electrophoretic mobility shift assays. RESULTS: The 32,612, 30,837 and 30,277 genes expressed at 5, 10 and 15 days postanthesis (dpa) were grouped into 19 distinct coexpression modules, and 988 genes in the MEblack module were enriched in the cell wall process and exhibited significant associations with FL. A total of 20 FL-QTLs were identified, each explaining 3.34-16.04 % of the phenotypic variance in the FL. Furthermore, several FL-QTLs contained 15 genes that were differentially expressed in the MEblack module including the tubulin beta gene (TUB5). Compared with the wild type, the overexpression of GhTUB5 and GbTUB5 in Arabidopsis suppressed root cell length but promoted cellulose synthesis. Knockout of GhTUB5 resulted in longer fiber lines. Protein-based experiments revealed that GhTUB5 interacts with GhZFP6. Additionally, GhTUB5 was directly activated by GhHD-ZIP7, a homeobox-leucine zipper transcription factor, and its paralogous gene was previously reported to mediate fiber elongation. CONCLUSION: This study opens a new avenue to dissect functional mechanism of cotton fiber elongation. Our findings provide some molecular details on how GhTUB5 mediates the FL phenotype in cotton.

Identification and characterization of circular RNAs involved in the fertility stability of cotton CMS-D2 restorer line under heat stress.

BACKGROUND: As a vital type of noncoding RNAs, circular RNAs (circRNAs) play important roles in plant growth and development and stress response. However, little is known about the biological roles of circRNAs in regulating the stability of male fertility restoration for cytoplasmic male sterility (CMS) conditioned by Gossypium harknessii cytoplasm (CMS-D2) cotton under high-temperature (HT) stress. RESULTS: In this study, RNA-sequencing and bioinformatics analysis were performed on pollen grains of isonuclear alloplasmic near-isogenic restorer lines NH [N(Rf1rf1)] and SH [S(Rf1rf1)] with obvious differences in fertility stability under HT stress at two environments. A total of 967 circRNAs were identified, with 250 differentially expressed under HT stress. We confirmed the back-splicing sites of eight selected circRNAs using divergent primers and Sanger sequencing. Tissue-specific expression patterns of five differentially expressed circRNAs (DECs) were also verified by RT-PCR and qRT-PCR. Functional enrichment and metabolic pathway analysis revealed that the parental genes of DECs were significantly enriched in fertility-related biological processes such as pollen tube guidance and cell wall organization, as well as the Pentose and glucuronate interconversions, Steroid biosynthesis, and N-Glycan biosynthesis pathways. Moreover, we also constructed a putative circRNA-mediated competing endogenous RNA (ceRNA) network consisting of 21 DECs, eight predicted circRNA-binding miRNAs, and their corresponding 22 mRNA targets, especially the two ceRNA modules circRNA346-miR159a-MYB33 and circRNA484-miR319e-MYB33, which might play important biological roles in regulating pollen fertility stability of cotton CMS-D2 restorer line under HT stress. CONCLUSIONS: Through systematic analysis of the abundance, characteristics and expression patterns of circRNAs, as well as the potential functions of their parent genes, our findings suggested that circRNAs and their mediated ceRNA networks acted vital biological roles in cotton pollen development, and might be also essential regulators for fertility stability of CMS-D2 restorer line under heat stress. This study will open a new door for further unlocking complex regulatory mechanisms underpinning the fertility restoration stability for CMS-D2 in cotton.

Dose effects of restorer gene modulate pollen fertility in cotton CMS-D2 restorer lines via auxin signaling and flavonoid biosynthesis.

KEY MESSAGE: Dose effects of Rf1 gene regulated retrieval mechanism of pollen fertility for CMS-D2 cotton. Cytoplasmic male sterility conditioned by Gossypium harknessii cytoplasm (CMS-D2) is an economical pollination control system for producing hybrid cotton seeds compared to artificial and chemical emasculation methods. However, the unstable restoring ability of restorer lines is a main barrier in the large-scale application of "three-line" hybrid cotton in China. Our phenotypic investigation determined that the homozygous Rf1Rf1 allelic genotype had a stronger ability to generate fertile pollen than the heterozygous Rf1rf1 allelic genotype. To decipher the genetic mechanisms that control the differential levels of pollen fertility, an integrated metabolomic and transcriptomic analysis was performed at two environments using pollen grains of four cotton genotypes differing in Rf1 alleles or cytoplasm. Totally 5,391 differential metabolite features were detected, and 369 specific differential metabolites (DMs) were identified between homozygous and heterozygous Rf1 allelic genotypes with CMS-D2 cytoplasm. In addition, transcriptome analysis identified 2,490 differentially expressed genes (DEGs) and 96 unique hub DEGs with dynamic regulation in this comparative combination. Further integrated analyses revealed that several key DEGs and DMs involved in indole biosynthesis, flavonoid biosynthesis, and sugar metabolism had strong network linkage with fertility restoration. In vitro application of auxin analogue NAA and inhibitor Auxinole confirmed that over-activated auxin signaling might inhibit pollen development, whereas suppressing auxin signaling partially promoted pollen development in CMS-D2 cotton. Our results provide new insight into how the dosage effects of the Rf1 gene regulate pollen fertility of CMS-D2 cotton.

Comparative Transcriptome Profiling of CMS-D2 and CMS-D8 Systems Characterizes Fertility Restoration Genes Network in Upland Cotton.

Resolving the genetic basis of fertility restoration for cytoplasmic male sterility (CMS) can improve the efficiency of three-line hybrid breeding. However, the genetic determinants of male fertility restoration in cotton are still largely unknown. This study comprehensively compared the full-length transcripts of CMS-D2 and CMS-D8 systems to identify potential genes linked with fertility restorer genes Rf1 or Rf2. Target comparative analysis revealed a higher percentage of differential genes in each restorer line as compared to their corresponding sterile and maintainer lines. An array of genes with specific expression in the restorer line of CMS-D2 had functional annotations related to floral development and pathway enrichments in various secondary metabolites, while specifically expressed genes in the CMS-D8 restorer line showed functional annotations related to anther development and pathway enrichment in the biosynthesis of secondary metabolites. Further analysis identified potentially key genes located in the target region of fertility restorer genes Rf1 or Rf2. In particular, Ghir_D05G032450 can be the candidate gene related to restorer gene Rf1, and Ghir_D05G035690 can be the candidate gene associated with restorer gene Rf2. Further gene expression validation with qRT-PCR confirmed the accuracy of our results. Our findings provide useful insights into decoding the potential regulatory network that retrieves pollen fertility in cotton and will help to further reveal the differences in the genetic basis of fertility restoration for two CMS systems.

Development of mitochondrial simple sequence repeat markers to simultaneously distinguish cytoplasmic male sterile sources in cotton.

Deleterious effects on anther development and main economy traits caused by sterile genes or cytoplasms are one of the important genetic characteristics of cytoplasmic male sterility (CMS) systems in cotton, which severely hinder the large-scale application of "three-line" hybrids in production. Therefore, distinct characterization of each cytoplasmic type is mandatory to improve the breeding efficiency of cotton hybrids. In this study, four isonuclear-alloplasmic cotton male sterile lines with G. hirsutum (CMS-(AD)1), G. barbadense (CMS-(AD)2), G. harknessii (CMS-D2), and G. trilobum (CMS-D8) cytoplasms were first created by multiple backcrosses with common genotype Shikang126. Then, 64 pairs of mitochondrial simple sequence repeat (mtSSR) markers were designed to explore the mitochondrial DNA diversities among four isonuclear-alloplasmic cotton male sterile lines, and a total of nine pairs of polymorphic mtSSR molecular markers were successfully developed. Polymorphism analysis indicated that mtSSR59 marker correlated to the atp1 gene could effectively divide the CMS-D2, CMS-(AD)1, and CMS-(AD)2 in one category while the CMS-D8 in another category. Further cytological observation and determination of ATP contents also confirmed the accurate classification of CMS-D2 and CMS-D8 lines. Moreover, the mtSSR59 marker was successfully applied in the marker-assisted selection (MAS) for breeding new male sterile lines and precise differentiation or purity identification of different CMS-based "three-line" and conventional cotton hybrids. This study provides new technical measures for classifying various cytoplasmic sterile lines, and our results will significantly improve the efficiency of there-line hybrid breeding in cotton.

Analysis of the MIR396 gene family and the role of MIR396b in regulating fiber length in cotton.

Cotton fiber is one of the most important natural raw materials in the world textile industry. Improving fiber yield and quality has always been the main goal. MicroRNAs, as typical small noncoding RNAs, could affect fiber length during different stages of fiber development. Based on differentially expressed microRNA in the two interspecific backcross inbred lines (BILs) with a significant difference in fiber length, we identified the miR396 gene family in the two tetraploid cotton genomes and found MIR396b_D13 as the functional precursor to produce mature miR396 during the fiber elongation stage. Among 46 target genes regulated by miR396b, the GROWTH-REGULATING FACTOR 5 gene (GRF5, Gh_A10G0492) had a differential expression level in the two BILs during fiber elongation stage. The expression patterns indicated that the miR396b-GRF5 regulatory module has a critical role in fiber development. Furthermore, virus-induced gene silencing (VIGS) of miR396b significantly produced longer fiber than the wild type, and the expression level of GRF5 showed the reverse trends of the miR396b expression level. The analysis of co-expression network for the GRF5 gene suggested that a cytochrome P450 gene functions as an allene oxide synthase (Gh_D06G0089, AOS), which plays a critical role in jasmonate biosynthetic pathway. In conclusion, our results revealed that the miR396b-GRF5 module has a critical role in fiber development. These findings provide a molecular foundation for fiber quality improvement in the future.

Integrated analysis of metabolome and transcriptome reveals the cytoplasmic effects of CMS-D2 on pollen fertility resulting from disrupted lipid metabolism.

Using cytoplasmic male sterility of Gossypium harknesii (CMS-D2) is an economical and effective method to produce cotton hybrids. However, the detrimental cytoplasmic effects of CMS-D2 on pollen fertility and fiber yields greatly limit the further development of three-line hybrid cotton in China. In this study, an integrated non-targeted metabolomics and transcriptome analysis was performed on mature pollens of maintainer line NB, isonuclear alloplasmic near-isogenic restorer lines NH and SH under two environments. A total of 820 metabolites were obtained, of which lipids and lipid-like molecules were the most, followed by organic acids derivatives, phenylpropanoids, and polyketides. Transcriptome analysis revealed significantly more differentially expressed genes (DEGs) in SH versus NH both in Anyang and Jiujiang, and most of the DEGs were significantly upregulated. Further KEGG analysis showed that most DEGs were enriched in the biosynthesis of unsaturated fatty acids, phenylalanine metabolism, and phagosome. Based on the weighted gene co-expression network analysis, totally 74 hub genes were also identified, of which three transcription factors, i.e., WRKY22, WRKY53, and ARF18 were significantly upregulated in SH and may play a negative regulatory role in pollen development by directly or indirectly regulating the jasmonic acid synthesis and signal transduction. Moreover, we found that the negative effects of CMS-D2 cytoplasm on pollen fertility were mainly due to disturbed lipid metabolism, especially the metabolic balance of unsaturated fatty acids, ultimately resulting in the decline of pollen fertility. Meanwhile, in the presence of CMS-D2 sterile cytoplasm, the cytoplasmic-nucleus interaction effects generated a substantial quantity of flavonoids involved in the fertility restoration process. This study preliminarily clarified some of the reasons for the negative effects of CMS-D2 cytoplasm on pollen fertility, and our results will provide an important theoretical reference for further breeding and improvement of three-line hybrid cotton in the future.

Transcript Complexity and New Insights of Restorer Line in CMS-D8 Cotton Through Full-Length Transcriptomic Analysis.

Hybrid utilization has proficiently increased crop production worldwide. The cytoplasmic male sterility (CMS) system has emerged as an efficient tool for commercial hybrid cotton seed production. The restorer line with dominant Rf2 gene can restore the fertility of the CMS-D8 sterile line. However, the molecular mechanism of fertility restoration remains unclear in CMS-D8 cotton that limits wider utilization of three-line hybrid breeding. In our study, the Pacific Biosciences (PacBio) Iso-Seq technology was applied to understand fertility restoration mechanism of CMS-D8 cotton. In total, 228,106 full-length non-chimeric transcriptome sequences were obtained from anthers of developing flowering buds. The analysis results identified 3,174 novel isoforms, 2,597 novel gene loci, 652 long non-coding RNAs predicted from novel isoforms, 7,234 alternative splicing events, 114 fusion transcripts, and 1,667 genes with alternative polyadenylation. Specially, two novel genes associated with restoration function, Ghir_D05.742.1 and m64033_190821_201011/21103726/ccs were identified and showed significant higher levels of expression in restorer line than sterile and maintainer lines. Our comparative full-length transcriptome analysis provides new insights into the molecular function of Rf2 fertility restorer gene. The results of this study offer a platform for fertility restoration candidate gene discovery in CMS-D8 cotton.

Homoeolog gene expression analysis reveals novel expression biases in upland hybrid cotton under intraspecific hybridization.

Hybridization is useful to enhance the yield potential of agronomic crops in the world. Cotton has genome doubling due to the allotetraploid process and hybridization in coordination with duplicated genome can produce more yield and adaptability. Therefore, the expression of homoeologous gene pairs between hybrids and inbred parents is vital to characterize the genetic source of heterosis in cotton. Investigation results of homoeolog gene pairs between two contrasting hybrids and their respective inbred parents identified 36853 homoeolog genes in hybrids. It was observed both high and low hybrids had similar trends in homoeolog gene expression patterns in each tissue under study. An average of 96% of homoeolog genes had no biased expression and their expressions were derived from the equal contribution of both parents. Besides, very few homoeolog genes (an average of 1%) showed no biased or novel expression in both hybrids. The functional analysis described secondary metabolic pathways had a majority of novel biased homoeolog genes in hybrids. These results contribute preliminary knowledge about how hybridization affects expression patterns of homoeolog gene pairs in upland cotton hybrids. Our study also highlights the functional genomics of metabolic genes to explore the genetic mechanism of heterosis in cotton.

Surface-enhanced Raman spectroscopy (SERS) for monitoring colistin-resistant and susceptible E. coli strains.

The emergence of drug-resistant bacteria is a precarious global health concern. In this study, surface-enhanced Raman spectroscopy (SERS) is used to characterize colistin-resistant and susceptible E. coli strains based on their distinguished SERS spectral features for the development of rapid and cost-effective detection and differentiation methods. For this purpose, three colistin-resistant and three colistin susceptible E. coli strains were analyzed by comparing their SERS spectral signatures. Moreover, multivariate data analysis techniques including Principal component analysis (PCA) and Partial Least Squares-Discriminant Analysis (PLS-DA) were used to examine the SERS spectral data of colistin-resistant and susceptible strains. PCA technique was employed for differentiating colistin susceptible and resistant E.coli strains due to alteration in biochemical compositions of the bacterial cell. PLS-DA is employed on SERS spectral data sets for discrimination of these resistant and susceptible E. coli strains with 100% specificity, 100% accuracy, 99.8% sensitivity, and 86% area under receiver operating characteristics (AUROC) curve.

Raman spectroscopic characterization of selenium N-heterocyclic carbene compounds.

In this study, Raman spectroscopy is employed to analyze and characterize two salts (N-heterocyclic carbene) and their respective selenium N-heterocyclic carbene compounds. The features observed as differences among Raman spectral data of two different N-heterocyclic carbene salts are called Salt-I and Salt-II and their respective Se compounds, called Compound-I & Compound-II, are used to confirm the formation covalent bond between Se atom carbon atom of carbene. Enhancement in peak intensities and shifting of peak positions is directly related with compound formation. Raman spectral data provide a detail information about bond formation, chemical and structural differences between salts and compounds. The observed Raman spectral features of both salts and compounds are in consistent with computationally calculated Raman spectral features. Raman spectral features of each salt and its respective compound was further analyzed with principal component analysis, which was found helpful for differentiating each salt from its respective compound.

Exogenously Applied Gibberellic Acid Enhances Growth and Salinity Stress Tolerance of Maize through Modulating the Morpho-Physiological, Biochemical and Molecular Attributes.

Soil salinity is the major limiting factor restricting plant growth and development. Little is known about the comparative and combined effects of gibberellic acid (GA3) seed priming and foliar application on maize under salt stress. The current study determined the impact of different application methods of GA3 on morpho-physiological, biochemical and molecular responses of maize seedlings under three salinity stress treatments (no salinity, moderate salinity-6 dS m-1, and severe salinity-12 dS m-1). The GA3 treatments consisted of control, hydro-priming (HP), water foliar spray (WFS), HP + WFS, seed priming with GA3 (GA3P, 100 mg L-1), foliar spray with GA3 (GA3FS, 100ppm) and GA3P + GA3FS. Salt stress particularly at 12 dS m-1 reduced the length of shoots and roots, fresh and dry weights, chlorophyll, and carotenoid contents, K+ ion accumulation and activities of antioxidant enzymes, while enhanced the oxidative damage and accumulation of the Na+ ion in maize plants. Nevertheless, the application of GA3 improved maize growth, reduced oxidative stress, and increased the antioxidant enzymes activities, antioxidant genes expression, and K+ ion concentration under salt stress. Compared with control, the GA3P + GA3FS recorded the highest increase in roots and shoots length (19-37%), roots fresh and dry weights (31-43%), shoots fresh and dry weights (31-47%), chlorophyll content (21-70%), antioxidant enzymes activities (73.03-150.74%), total soluble protein (13.05%), K+ concentration (13-23%) and antioxidants genes expression levels under different salinity levels. This treatment also reduced the H2O2 content, and Na+ ion concentration. These results indicated that GA3P + GA3FS could be used as an effective tool for improving the maize growth and development, and reducing the oxidative stress in salt-contaminated soils.

Comparative transcriptome analysis between inbred and hybrids reveals molecular insights into yield heterosis of upland cotton.

BACKGROUND: Utilization of heterosis has greatly improved the productivity of many crops worldwide. Understanding the potential molecular mechanism about how hybridization produces superior yield in upland cotton is critical for efficient breeding programs. RESULTS: In this study, high, medium, and low hybrids varying in the level of yield heterosis were screened based on field experimentation of different years and locations. Phenotypically, high hybrid produced a mean of 14% more seed cotton yield than its better parent. Whole-genome RNA sequencing of these hybrids and their four inbred parents was performed using different tissues of the squaring stage. Comparative transcriptomic differences in each hybrid parent triad revealed a higher percentage of differentially expressed genes (DEGs) in each tissue. Expression level dominance analysis identified majority of hybrids DEGs were biased towards parent like expressions. An array of DEGs involved in ATP and protein binding, membrane, cell wall, mitochondrion, and protein phosphorylation had more functional annotations in hybrids. Sugar metabolic and plant hormone signal transduction pathways were most enriched in each hybrid. Further, these two pathways had most mapped DEGs on known seed cotton yield QTLs. Integration of transcriptome, QTLs, and gene co-expression network analysis discovered genes Gh_A03G1024, Gh_D08G1440, Gh_A08G2210, Gh_A12G2183, Gh_D07G1312, Gh_D08G1467, Gh_A03G0889, Gh_A08G2199, and Gh_D05G0202 displayed a complex regulatory network of many interconnected genes. qRT-PCR of these DEGs was performed to ensure the accuracy of RNA-Seq data. CONCLUSIONS: Through genome-wide comparative transcriptome analysis, the current study identified nine key genes and pathways associated with biological process of yield heterosis in upland cotton. Our results and data resources provide novel insights and will be useful for dissecting the molecular mechanism of yield heterosis in cotton.

An Improved Rosin Transfer Process for the Reduction of Residue Particles for Graphene.

In this work, an improved rosin transfer process is initiated. An anisole coating is introduced based on the rosin transfer process to reduce the residue particles on the surface of transferred graphene. Rosin/graphene and anisole/rosin/graphene samples are handled without baking and with baking at different temperatures, i.e., 100 °C, 150 °C, and 200 °C. Atomic force microscopy (AFM) and Raman spectroscopy are employed to characterize the surface properties of transferred graphene. The removal of the protective rosin layer and anisole/rosin layers without baking is found to be more effective and beneficial compared to the conventional PMMA transfer process. Furthermore, better results in terms of reduced surface roughness and residue particles are accomplished by introducing anisole in the improved rosin transfer process. Uniform and low sheet resistance (Rsh) is also observed across transferred graphene using this improved process.

Comparative transcriptome analysis of inbred lines and contrasting hybrids reveals overdominance mediate early biomass vigor in hybrid cotton.

BACKGROUND: Heterosis breeding is the most useful method for yield increase around the globe. Heterosis is an intriguing process to develop superior offspring to either parent in the desired character. The biomass vigor produced during seedling emergence stage has a direct influence on yield heterosis in plants. Unfortunately, the genetic basis of early biomass vigor in cotton is poorly understood. RESULTS: Three stable performing F1 hybrids varying in yield heterosis named as high, medium and low hybrids with their inbred parents were used in this study. Phenotypically, these hybrids established noticeable biomass heterosis during the early stage of seedling growth in the field. Transcriptome analysis of root and leaf revealed that hybrids showed many differentially expressed genes (DEGs) relative to their parents, while the comparison of inbred parents showed limited number of DEGs indicating similarity in their genetic constitution. Further analysis indicated expression patterns of most DEGs were overdominant in both tissues of hybrids. According to GO results, functions of overdominance genes in leaf were enriched for chloroplast, membrane, and protein binding, whereas functions of overdominance genes in root were enriched for plasma membrane, extracellular region, and responses to stress. We found several genes of circadian rhythm pathway related to LATE ELONGATED HYPOCOTYL (LHY) showed downregulated overdominant expressions in both tissues of hybrids. In addition to circadian rhythm, several leaf genes related to Aux/IAA regulation, and many root genes involved in peroxidase activity also showed overdominant expressions in hybrids. Twelve genes involved in circadian rhythm plant were selected to perform qRT-PCR analysis to confirm the accuracy of RNA-seq results. CONCLUSIONS: Through genome-wide comparative transcriptome analysis, we strongly predict that overdominance at gene expression level plays a pivotal role in early biomass vigor of hybrids. The combinational contribution of circadian rhythm and other metabolic process may control vigorous growth in hybrids. Our result provides an important foundation for dissecting molecular mechanisms of biomass vigor in hybrid cotton.

Development and utilization of an InDel marker linked to the fertility restorer genes of CMS-D8 and CMS-D2 in cotton.

The cytoplasmic male sterility (CMS) system is a useful tool for commercial hybrid cotton seed production. Two main CMS systems, CMS-D8 and CMS-D2, have been recognized with Rf2 and Rf1 as the restorer genes, respectively. The development of molecular markers tightly linked with restorer genes can facilitate the breeding of restorer lines. In this study, the InDel-1892 marker was developed to distinguish Rf2 and Rf1 simultaneously. Sequence alignment implied that CMS-D8-Rf2 has a 32 bp insertion and that CMS-D2-Rf1 has a 186 bp insertion at the InDel-1892 locus. The codominant marker was co-segregated with Rf1 and Rf2. Hence, this marker can be used for tracing Rf1 and Rf2 simultaneously and identifying the allele status at the restorer gene locus. The results of this study will facilitate efficient marker-assisted selection for restorer lines and hybrids of CMS systems.

Single-base resolution methylome of cotton cytoplasmic male sterility system reveals epigenomic changes in response to high-temperature stress during anther development.

Anther development in flowering plants is highly sensitive to high-temperature (HT) stress. Understanding the potential epigenetic mechanism of anther infertility induced by HT stress in cotton (Gossypium hirsutum L.) is crucial for the effective use of genetic resources to guide plant breeding. Using the whole-genome bisulfite sequencing, we map cytosine methylation at single-base resolution across the whole genome of cotton anthers, and changes in the methylome of the cytoplasmic male sterility system associated with HT stress were analysed in two cotton lines with contrasting HT stress tolerance. The cotton anther genome was found to display approximately 31.6%, 68.7%, 61.8%, and 21.8% methylation across all sequenced C sites and in the CG, CHG, and CHH sequence contexts, respectively. In an integrated global methylome and transcriptome analysis, only promoter-unmethylated genes showed higher expression levels than promoter-methylated genes, whereas gene body methylation presented an obvious positive correlation with gene expression. The methylation profiles of transposable elements in cotton anthers were characterized, and more differentially methylated transposable elements were demethylated under HT stress. HT-induced promoter methylation changes led to the up-regulation of the mitochondrial respiratory chain enzyme-associated genes GhNDUS7, GhCOX6A, GhCX5B2, and GhATPBM, ultimately promoting a series of redox processes to form ATP for normal anther development under HT stress. In vitro application of the common DNA methylation inhibitor 5-azacytidine and accelerator methyl trifluoromethanesulfonate demonstrated that DNA demethylation promoted anther development, while increased methylation only partially inhibited anther development under HT stress.

Integrated Methylome and Transcriptome Analysis between the CMS-D2 Line ZBA and Its Maintainer Line ZB in Upland Cotton.

DNA methylation is an important epigenetic modification involved in multiple biological processes. Altered methylation patterns have been reported to be associated with male sterility in some plants, but their role in cotton cytoplasmic male sterility (CMS) remains unclear. Here, integrated methylome and transcriptome analyses were conducted between the CMS-D2 line ZBA and its near-isogenic maintainer line ZB in upland cotton. More methylated cytosine sites (mCs) and higher methylation levels (MLs) were found among the three sequence contexts in ZB compared to ZBA. A total of 4568 differentially methylated regions (DMRs) and 2096 differentially methylated genes (DMGs) were identified. Among the differentially expressed genes (DEGs) associated with DMRs (DMEGs), 396 genes were upregulated and 281 genes were downregulated. A bioinformatics analysis of these DMEGs showed that hyper-DEGs were significantly enriched in the "oxidative phosphorylation" pathway. Further qRT-PCR validation indicated that these hypermethylated genes (encoding the subunits of mitochondrial electron transport chain (ETC) complexes I and V) were all significantly upregulated in ZB. Our biochemical data revealed a higher extent of H2O2 production but a lower level of adenosine triphosphate (ATP) synthesis in CMS-D2 line ZBA. On the basis of the above results, we propose that disrupted DNA methylation in ZBA may disrupt the homeostasis of reactive oxygen species (ROS) production and ATP synthesis in mitochondria, triggering a burst of ROS that is transferred to the nucleus to initiate programmed cell death (PCD) prematurely, ultimately leading to microspore abortion. This study illustrates the important role of DNA methylation in cotton CMS.

Effects of Different Ion Irradiation on the Contact Resistance of Pd/Graphene Contacts.

The effect of ion-induced defects on graphene was studied to investigate the contact resistance of 40 nm palladium (Pd) contacting on graphene. The defect development was considered and analyzed by irradiating boron (B), carbon (C), nitrogen (N2), and argon (Ar) ions on as-transferred graphene before metallization. The bombardment energy was set at 1.5 keV and ion dose at 1 × 1014 ions/cm2. The defect yields under different ion irradiation conditions were examined by Raman spectroscopy. Although, dissolution process occurs spontaneously upon metal deposition, chemical reaction between metal and graphene is more pronounced at higher temperatures. The rapid thermal annealing (RTA) treatment was performed to improve the Pd/graphene contact after annealing at 450 °C, 500 °C, 550 °C, and 600 °C. The lowest contact resistance of 95.2 Ω-µm was achieved at 550 °C RTA with Ar ion irradiation. We have proved that ion irradiation significantly enhance the Pd/graphene contact instead of pd/pristine graphene contact. Therefore, in view of the contention of results ion induced defects before metallization plus the RTA served an excellent purpose to reduce the contact resistance.