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.

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.

Heat-responsive microRNAs participate in regulating the pollen fertility stability of CMS-D2 restorer line under high-temperature stress.

Anther development and pollen fertility of cytoplasmic male sterility (CMS) conditioned by Gossypium harknessii cytoplasm (CMS-D2) restorer lines are susceptible to continuous high-temperature (HT) stress in summer, which seriously hinders the large-scale application of "three-line" hybrids in production. Here, integrated small RNA, transcriptome, degradome, and hormone profiling was performed to explore the roles of microRNAs (miRNAs) in regulating fertility stability in mature pollens of isonuclear alloplasmic near-isogenic restorer lines NH and SH under HT stress at two environments. A total of 211 known and 248 novel miRNAs were identified, of which 159 were differentially expressed miRNAs (DEMs). Additionally, 45 DEMs in 39 miRNA clusters (PmCs) were also identified, and most highly expressed miRNAs were significantly induced in SH under extreme HT, especially four MIR482 and six MIR6300 family miRNAs. PmC28 was located in the fine-mapped interval of the Rf1 gene and contained two DEMs, gra-miR482_L-2R + 2 and gma-miR2118a-3p_R + 1_1ss18TG. Transcriptome sequencing identified 6281 differentially expressed genes, of which heat shock protein (HSP)-related genes, such as HSP70, HSP22, HSP18.5-C, HSP18.2 and HSP17.3-B, presented significantly reduced expression levels in SH under HT stress. Through integrating multi-omics data, we constructed a comprehensive molecular network of miRNA-mRNA-gene-KEGG containing 35 pairs of miRNA/target genes involved in regulating the pollen development in response to HT, among which the mtr-miR167a_R + 1, tcc-miR167c and ghr-miR390a, tcc-miR396c_L-1 and ghr-MIR169b-p3_1ss6AG regulated the pollen fertility by influencing ARF8 responsible for the auxin signal transduction, ascorbate and aldarate metabolism, and the sugar and lipid metabolism and transport pathways, respectively. Further combination with hormone analysis revealed that HT-induced jasmonic acid signaling could activate the expression of downstream auxin synthesis-related genes and cause excessive auxin accumulation, followed by a cascade of auxin signal transduction, ultimately resulting in pollen abortion. The results provide a new understanding of how heat-responsive miRNAs regulate the stability of fertility restoration for CMS-D2 cotton under heat stress.

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.

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.

Single-molecule real-time transcript sequencing of developing cotton anthers facilitates genome annotation and fertility restoration candidate gene discovery.

Heterosis refers to the superior phenotypes observed in hybrids. Cytoplasmic male sterility (CMS) system plays an important role in cotton heterosis utilization. However, the global gene expression patterns of CMS-D2 and its interaction with the restorer gene Rf1 remain unclear. Here, the full-length transcript sequencing was performed in anthers of the CMS-D2 restorer line using PacBio single-molecule real-time sequencing technology. Combining PacBio SMRT long-read isoforms and Illumina RNA-seq data, 107,066 isoforms from 44,338 loci were obtained, including 10,086 novel isoforms of novel genes and 66,419 new isoforms of known genes. Totally 56,572 alternative splicing (AS) events, 1146 lncRNAs, 61 fusion transcripts and 10,466 genes exhibited alternative polyadenylation (APA), and 60,995 novel isoforms with predicted open reading frames (ORFs) were further identified. Furthermore, the specifically expressed genes in restorer line were selected and confirmed by qRT-PCR. These findings provide a basis for upland cotton genome annotation and transcriptome research, and will help to reveal the molecular mechanism of interaction between Rf1 and CMS-D2 cytoplasm.

Physical mapping and InDel marker development for the restorer gene Rf2 in cytoplasmic male sterile CMS-D8 cotton.

BACKGROUND: Cytoplasmic male sterile (CMS) with cytoplasm from Gossypium Trilobum (D8) fails to produce functional pollen. It is useful for commercial hybrid cotton seed production. The restore line of CMS-D8 containing Rf2 gene can restore the fertility of the corresponding sterile line. This study combined the whole genome resequencing bulked segregant analysis (BSA) with high-throughput SNP genotyping to accelerate the physical mapping of Rf2 locus in CMS-D8 cotton. METHODS: The fertility of backcross population ((sterile line×restorer line)×maintainer line) comprising of 1623 individuals was investigated in the field. The fertile pool (100 plants with fertile phenotypes, F-pool) and the sterile pool (100 plants with sterile phenotypes, S-pool) were constructed for BSA resequencing. The selection of 24 single nucleotide polymorphisms (SNP) through high-throughput genotyping and the development insertion and deletion (InDel) markers were conducted to narrow down the candidate interval. The pentapeptide repeat (PPR) family genes and upregulated genes in restore line in the candidate interval were analysed by qRT-PCR. RESULTS: The fertility investigation results showed that fertile and sterile separation ratio was consistent with 1:1. BSA resequencing technology, high-throughput SNP genotyping, and InDel markers were used to identify Rf2 locus on candidate interval of 1.48 Mb on chromosome D05. Furthermore, it was quantified in this experiment that InDel markers co-segregated with Rf2 enhanced the selection of the restorer line. The qRT-PCR analysis revealed PPR family gene Gh_D05G3391 located in candidate interval had significantly lower expression than sterile and maintainer lines. In addition, utilization of anther RNA-Seq data of CMS-D8 identified that the expression level of Gh_D05G3374 encoding NB-ARC domain-containing disease resistance protein in restorer lines was significantly higher than that in sterile and maintainer lines. CONCLUSIONS: This study not only enabled us to precisely locate the restore gene Rf2 but also evaluated the utilization of InDel markers for marker assisted selection in the CMS-D8 Rf2 cotton breeding line. The results of this study provide an important foundation for further studies on the mapping and cloning of restorer genes.

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.

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.

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.

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.

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.

Transcriptome Analysis Implicates Involvement of Long Noncoding RNAs in Cytoplasmic Male Sterility and Fertility Restoration in Cotton.

The cytoplasmic male sterility (CMS)/restorer-of-fertility system is an important tool to exploit heterosis during commercially hybrid seed production. The importance of long noncoding RNAs (lncRNAs) in plant development is recognized, but few analyses of lncRNAs during anther development of three-line hybrid cotton (CMS-D2 line A, maintainer line B, restorer-of-fertility line R) have been reported. Here, we performed transcriptome sequencing during anther development in three-line hybrid cotton. A total of 80,695 lncRNAs were identified, in which 43,347 and 44,739 lncRNAs were differentially expressed in A-B and A-R comparisons, respectively. These lncRNAs represent functional candidates involved in CMS and fertility restoration. GO analysis indicated that cellular hormone metabolic processes and oxidation-reduction reaction processes might be involved in CMS, and cellular component morphogenesis and small molecular biosynthetic processes might participate in fertility restoration. Additionally, 63 lncRNAs were identified as putative precursors of 35 miRNAs, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed a similar expression pattern to RNA-seq data. Furthermore, construction of lncRNA regulatory networks indicated that several miRNA-lncRNA-mRNA networks might be involved in CMS and fertility restoration. Our findings provide systematic identification of lncRNAs during anther development and lays a solid foundation for the regulatory mechanisms and utilization in hybrid cotton breeding.

A combined small RNA and transcriptome sequencing analysis reveal regulatory roles of miRNAs during anther development of Upland cotton carrying cytoplasmic male sterile Gossypium harknessii (D2) cytoplasm.

BACKGROUND: Cytoplasmic male sterility (CMS) in flowering plants is usually caused by incompatibility between mitochondrial and nuclear genomes, and can be restored by nuclear genes known as restorer-of-fertility (Rf). Although the CMS/Rf system is useful and convenient for economic production of commercial hybrid seed, the molecular mechanisms of CMS occurrence and fertility restoration in cotton are unclear. RESULTS: Here, a combined small RNA and transcriptome sequencing analysis was performed on floral buds at the meiosis stage in three-line hybrid cotton system, and differentially expressed microRNAs (DEMs) and their target genes were identified and further analyzed for a possible involvement in CMS and fertility restoration. Totally 10 and 30 differentially expressed miRNA-target gene pairs were identified in A-B and A-R comparison group, respectively. A putative regulatory network of CMS occurrence and fertility restoration-related miRNA-target pairs during anther development were then constructed. The RLM-RACE analysis showed that gra-miR7505b regulates a PPR gene (Gh_D05G3392) by cleaving precisely at the 643 nt and 748 nt sites. The further analysis indicated that the sequence variation in the binding regions of Gh_D05G3392 and Gh_D05G3356 may cause a lower cleavage efficiency of the PPR genes by miR7505b and miR7505 in R line, respectively, leading to the up-regulation of the PPR genes and fertility restoration. These results have established their genetic involvement in fertility restoration in the CMS-D2 system. CONCLUSION: Our combined miRNA and transcriptome analysis in three-line hybrid cotton system provides new insights into the molecular mechanisms of CMS occurrence and fertility restoration, which will contribute to further hybrid breeding in cotton.

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.

A genome-wide identification and analysis of the DYW-deaminase genes in the pentatricopeptide repeat gene family in cotton (Gossypium spp.).

The RNA editing occurring in plant organellar genomes mainly involves the change of cytidine to uridine. This process involves a deamination reaction, with cytidine deaminase as the catalyst. Pentatricopeptide repeat (PPR) proteins with a C-terminal DYW domain are reportedly associated with cytidine deamination, similar to members of the deaminase superfamily. PPR genes are involved in many cellular functions and biological processes including fertility restoration to cytoplasmic male sterility (CMS) in plants. In this study, we identified 227 and 211 DYW deaminase-coding PPR genes for the cultivated tetraploid cotton species G. hirsutum and G. barbadense (2n = 4x = 52), respectively, as well as 126 and 97 DYW deaminase-coding PPR genes in the ancestral diploid species G. raimondii and G. arboreum (2n = 26), respectively. The 227 G. hirsutum PPR genes were predicted to encode 52-2016 amino acids, 203 of which were mapped onto 26 chromosomes. Most DYW deaminase genes lacked introns, and their proteins were predicted to target the mitochondria or chloroplasts. Additionally, the DYW domain differed from the complete DYW deaminase domain, which contained part of the E domain and the entire E+ domain. The types and number of DYW tripeptides may have been influenced by evolutionary processes, with some tripeptides being lost. Furthermore, a gene ontology analysis revealed that DYW deaminase functions were mainly related to binding as well as hydrolase and transferase activities. The G. hirsutum DYW deaminase expression profiles varied among different cotton tissues and developmental stages, and no differentially expressed DYW deaminase-coding PPRs were directly associated with the male sterility and restoration in the CMS-D2 system. Our current study provides an important piece of information regarding the structural and evolutionary characteristics of Gossypium DYW-containing PPR genes coding for deaminases and will be useful for characterizing the DYW deaminase gene family in cotton biology and breeding.