High quality RNA extraction protocol for polyphenolics-rich Cotton tissue.

The extraction of high-quality RNA from cotton (Gossypium spp.) is challenging because of the presence of high polyphenolics, polysaccharides, quinones, and other secondary metabolites. A high-throughput RNA extraction protocol is a prerequisite. This Triton-X-100-based RNA extraction method utilizes Polyvinyl pyrrolidone polymer (PVPP) treatment which efficiently removes phenolics, and the application of Lithium chloride (LiCl) has been found that successfully precipitated the high-quality RNA from cotton tissue. Cytoplasmic male sterility (CMS) is a maternally inherited trait associated with specific mitochondrial genome rearrangements or mutations. The suitability of RNA extracted from Cotton CMS lines was assessed. cDNA was synthesized from RNA and assayed for mitochondrial genes (cox3, nad3, nad9) associated with male sterility. This paper discuss the advantages and limitation of this protocol over existing protocol for RNA extraction for polyphenolics-rich plant tissue.

A unique C-terminal domain contributes to the molecular function of Restorer-of-fertility proteins in plant mitochondria.

Restorer-of-fertility (Rf) genes encode pentatricopeptide repeat (PPR) proteins that are targeted to mitochondria where they specifically bind to transcripts that induce cytoplasmic male sterility and repress their expression. In searching for a molecular signature unique to this class of proteins, we found that a majority of known Rf proteins have a distinct domain, which we called RfCTD (Restorer-of-fertility C-terminal domain), and its presence correlates with the ability to induce cleavage of the mitochondrial RNA target. A screen of 219 angiosperm genomes from 123 genera using a sequence profile that can quickly and accurately identify RfCTD sequences revealed considerable variation in RFL/RfCTD gene numbers across flowering plants. We observed that plant genera with bisexual flowers have significantly higher numbers of RFL genes compared to those with unisexual flowers, consistent with a role of these proteins in restoration of male fertility. We show that removing the RfCTD from the RFL protein RNA PROCESSING FACTOR 2-nad6 prevented cleavage of its RNA target, the nad6 transcript, in Arabidopsis thaliana mitochondria. We provide a simple way of identifying putative Rf candidates in genome sequences, new insights into the molecular mode of action of Rf proteins and the evolution of fertility restoration in flowering plants.

Male sterility and hybrid breeding in soybean.

Hybrid breeding can help us to meet the challenge of feeding a growing world population with limited agricultural land. The demand for soybean is expected to grow; however, the hybrid soybean is still in the process of commercialization even though considerable progress has been made in soybean genome and genetic studies in recent years. Here, we summarize recent advances in male sterility-based breeding programs and the current status of hybrid soybean breeding. A number of male-sterile lines with cytoplasmic male sterility (CMS), genic-controlled photoperiod/thermo-sensitive male sterility, and stable nuclear male sterility (GMS) have been identified in soybean. More than 40 hybrid soybean varieties have been bred using the CMS three-line hybrid system and the cultivation of hybrid soybean is still under way. The key to accelerating hybrid soybean breeding is to increase the out-crossing rate in an economical way. This review outlines current problems with the hybrid soybean breeding systems and explores the current efforts to make the hybrid soybean a commercial success.

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.

Integrated Methylome and Transcriptome Analysis Provides Insights into the DNA Methylation Underlying the Mechanism of Cytoplasmic Male Sterility in Kenaf (Hibiscus cannabinus L.).

Cytoplasmic male sterility (CMS) is widely exploited in hybrid seed production. Kenaf is an important fiber crop with high heterosis. The molecular mechanism of kenaf CMS remains unclear, particularly in terms of DNA methylation. Here, using the anthers of a kenaf CMS line (P3A) and its maintainer line (P3B), comparative physiological, DNA methylation, and transcriptome analyses were performed. The results showed that P3A had considerably lower levels of IAA, ABA, photosynthetic products and ATP contents than P3B. DNA methylome analysis revealed 650 differentially methylated genes (DMGs) with 313 up- and 337 down methylated, and transcriptome analysis revealed 1788 differentially expressed genes (DEGs) with 558 up- and 1230 downregulated genes in P3A compared with P3B. Moreover, 45 genes were characterized as both DEGs and DMGs, including AUX,CYP, BGL3B, SUS6, AGL30 and MYB21. Many DEGs may be regulated by related DMGs based on methylome and transcriptome studies. These DEGs were involved in carbon metabolism, plant hormone signal transduction, the TCA cycle and the MAPK signaling pathway and were shown to be important for CMS in kenaf. These results provide new insights into the epigenetic mechanism of CMS in kenaf and other crops.

Comparison of Mitochondrial Genomes between a Cytoplasmic Male-Sterile Line and Its Restorer Line for Identifying Candidate CMS Genes in Gossypium hirsutum.

As the core of heterosis utilization, cytoplasmic male sterility (CMS) has been widely used in hybrid seed production. Previous studies have shown that CMS is always closely related to the altered programming of mitochondrial genes. To explore candidate CMS genes in cotton (Gossypium hirsutum), sequencing and de novo assembly were performed on the mitochondrial genome of the G. hirsutum CMS line SI3A, with G. harknessii CMS-D2 cytoplasm, and the corresponding G. hirsutum restorer line 0-613-2R. Remarkable variations in genome structure and gene transcripts were detected. The mitochondrial genome of SI3A has three circle molecules, including one main circle and two sub-circles, while 0-613-2R only has one. RNA-seq and RT-qPCR analysis proved that orf606a and orf109a, which have a chimeric structure and transmembrane domain, were highly expressed in abortive anthers of SI3A. In addition, comparative analysis of RNA-seq and full-length transcripts revealed the complex I gene nad4 to be expressed at a lower level in SI3A than in its restorer and that it featured an intron retention splicing pattern. These two novel chimeric ORFs and nad4 are potential candidates that confer CMS character in SI3A. This study provides new insight into the molecular basis of the nuclear-cytoplasmic interaction mechanism, and that putative CMS genes might be important sources for future precise design cross-breeding of cotton.

Comparative analysis of the complete mitochondrial genome sequences and anther development cytology between maintainer and Ogura-type cytoplasm male-sterile cabbage (B. oleracea Var. capitata).

BACKGROUND: Cytoplasmic male sterility (CMS) has been widely used for commercial F1 hybrid seeds production. CMS is primarily caused by chimeric genes in mitochondrial genomes. However, which specific stages of anther development in cabbage are affected by the chimeric genes remain unclear. RESULTS: In the present study, the complete mitochondrial genomes were sequenced and assembled for the maintainer and Ogura CMS cabbage lines. The genome size of the maintainer and Ogura CMS cabbage are 219,962 bp and 236,648 bp, respectively. There are 67 and 69 unknown function ORFs identified in the maintainer and Ogura CMS cabbage mitochondrial genomes, respectively. Four orfs, orf102a, orf122b, orf138a and orf154a were specifically identified in the Ogura CMS mitochondrial genome, which were likely generated by recombination with Ogura type radish during breeding process. Among them, ORF138a and ORF154a possessed a transmembrane structure, and orf138a was co-transcribed with the atp8 and trnfM genes. orf154a is partially homologous to the ATP synthase subunit 1 (atpA) gene. Both these genes were likely responsible for the CMS phenotype. In addition, cytological sections showed that the abnormal proliferation of tapetal cells might be the immediate cause of cytoplasmic male-sterility in Ogura CMS cabbage lines. RNA-seq results showed that orf138a and orf154a in Ogura CMS might influence transcript levels of genes in energy metabolic pathways. CONCLUSIONS: The presence of orf138a and orf154a lead to increased of ATPase activity and ATP content by affecting the transcript levels of genes in energy metabolic pathways, which could provide more energy for the abnormal proliferation of tapetal cells. Our data provides new insights into cytoplasmic male-sterility from whole mitochondrial genomes, cytology of anther development and transcriptome data.

A comprehensive integrated transcriptome and metabolome analyses to reveal key genes and essential metabolic pathways involved in CMS in kenaf.

KEY MESSAGE: Numbers of critical genes and pathways were found from the levels of transcriptome and metabolome, which were useful information for understanding of kenaf CMS mechanism. Cytoplasmic male sterility (CMS) is a maternally inherited trait in higher plants that leads to the inability to produce or release functional pollen. However, there is lack of comprehensive studies to reveal the molecular basis of CMS occurrence in kenaf. Herein, we performed transcriptome and UPLC-MS-based metabolome analyses in the anthers of a CMS (UG93A) and its maintainer (UG93B) to sort out essential genes and metabolites responding to CMS in kenaf. Transcriptome characterized 7769 differentially expressed genes (DEGs) between these two materials, and pathway enrichment analysis indicated that these DEGs were involved mainly in pentose and glucuronate interconversions, starch and sucrose metabolism, taurine and hypotaurine metabolism. In the metabolome assay, a total of 116 significantly different metabolites (SDMs) were identified between the CMS and its maintainer line, and these SDMs were involved in eight KEGG pathways, including flavone and flavonol biosynthesis, glycerophospholipid metabolism, flavonoid biosynthesis, glycosylphosphatidylinositol-anchor biosynthesi. Integrated analyses of transcriptome and metabolome showed that 50 genes had strong correlation coefficient values (R2 > 0.9) with ten metabolites enriched in six pathways; notably, most genes and metabolites of flavonoid biosynthesis pathways and flavone and flavonol biosynthesis pathways involved in flavonoids biosynthetic pathways were downregulated in CMS compared to those in maintainer. Taken together, the decreased accumulation of flavonoids resulted from the compromised biosynthesis pathways coupled with energy deficiency in the anthers may contribute largely to CMS in UG93A of kenaf.

Complete Mitochondrial Genome Sequence and Identification of a Candidate Gene Responsible for Cytoplasmic Male Sterility in Celery (Apium graveolens L.).

Celery (Apium graveolens L.) is an important leafy vegetable worldwide. The development of F1 hybrids in celery is highly dependent on cytoplasmic male sterility (CMS) because emasculation is difficult. In this study, we first report a celery CMS, which was found in a high-generation inbred line population of the Chinese celery "tanzhixiangqin". Comparative analysis, following sequencing and assembly of the complete mitochondrial genome sequences for this celery CMS line and its maintainer line, revealed that there are 21 unique regions in the celery CMS line and these unique regions contain 15 ORFs. Among these ORFs, only orf768a is a chimeric gene, consisting of 1497 bp sequences of the cox1 gene and 810 bp unidentified sequences located in the unique region, and the predicted protein product of orf768a possesses 11 transmembrane domains. In summary, the results of this study indicate that orf768a is likely to be a strong candidate gene for CMS induction in celery. In addition, orf768a can be a co-segregate marker, which can be used to screen CMS in celery.

Complete Plastid and Mitochondrial Genomes of Aeginetia indica Reveal Intracellular Gene Transfer (IGT), Horizontal Gene Transfer (HGT), and Cytoplasmic Male Sterility (CMS).

Orobanchaceae have become a model group for studies on the evolution of parasitic flowering plants, and Aeginetia indica, a holoparasitic plant, is a member of this family. In this study, we assembled the complete chloroplast and mitochondrial genomes of A. indica. The chloroplast and mitochondrial genomes were 56,381 bp and 401,628 bp long, respectively. The chloroplast genome of A. indica shows massive plastid genes and the loss of one IR (inverted repeat). A comparison of the A. indica chloroplast genome sequence with that of a previous study demonstrated that the two chloroplast genomes encode a similar number of proteins (except atpH) but differ greatly in length. The A. indica mitochondrial genome has 53 genes, including 35 protein-coding genes (34 native mitochondrial genes and one chloroplast gene), 15 tRNA (11 native mitochondrial genes and four chloroplast genes) genes, and three rRNA genes. Evidence for intracellular gene transfer (IGT) and horizontal gene transfer (HGT) was obtained for plastid and mitochondrial genomes. ψndhB and ψcemA in the A. indica mitogenome were transferred from the plastid genome of A. indica. The atpH gene in the plastid of A. indica was transferred from another plastid angiosperm plastid and the atpI gene in mitogenome A. indica was transferred from a host plant like Miscanthus siensis. Cox2 (orf43) encodes proteins containing a membrane domain, making ORF (Open Reading Frame) the most likely candidate gene for CMS development in A. indica.

Genomic Scan of Male Fertility Restoration Genes in a 'Gülzow' Type Hybrid Breeding System of Rye (Secale cereale L.).

Efficient and stable restoration of male fertility (Rf) is a prerequisite for large-scale hybrid seed production but remains an inherent issue in the predominant fertility control system of rye (Secale cereale L.). The 'Gülzow' (G)-type cytoplasmic male sterility (CMS) system in hybrid rye breeding exhibits a superior Rf. While having received little scientific attention, one major G-type Rf gene has been identified on 4RL (Rfg1) and two minor genes on 3R (Rfg2) and 6R (Rfg3) chromosomes. Here, we report a comprehensive investigation of the genetics underlying restoration of male fertility in a large G-type CMS breeding system using recent advents in rye genomic resources. This includes: (I) genome-wide association studies (GWAS) on G-type germplasm; (II) GWAS on a biparental mapping population; and (III) an RNA sequence study to investigate the expression of genes residing in Rf-associated regions in G-type rye hybrids. Our findings provide compelling evidence of a novel major G-type non-PPR Rf gene on the 3RL chromosome belonging to the mitochondrial transcription termination factor gene family. We provisionally denote the identified novel Rf gene on 3RL RfNOS1. The discovery made in this study is distinct from known P- and C-type systems in rye as well as recognized CMS systems in barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.). We believe this study constitutes a stepping stone towards understanding the restoration of male fertility in the G-type CMS system and potential resources for addressing the inherent issues of the P-type system.

A genome-wide analysis of pentatricopeptide repeat (PPR) protein-encoding genes in four Gossypium species with an emphasis on their expression in floral buds, ovules, and fibers in upland cotton.

Cotton is the most important natural fiber used in textiles. Breeding for "three-lines", i.e., cytoplasmic male sterility (CMS)-based sterile (A), maintainer (B), and restorer (R) line, is a promising approach to harness hybrid vigor in cotton. Pentatricopeptide repeat (PPR) protein-encoding genes play an important role in plant growth and development including restoration of CMS plants to male fertility. However, PPRs, especially those contributing to CMS and fiber development, remain largely unknown in cotton. In this study, a genome-wide identification and characterization of PPR gene family in four Gossypium species with genome sequences (G. arboreum, G. raimondii, G. hirsutum, and G. barbadense) were performed, and expressed PPR genes in developing floral buds, ovules, and fibers were compared to identify possible PPRs related to CMS restoration and fiber development. A total of 539, 558, 1032, and 1055 PPRs were predicted in the above four species, respectively, which were further mapped to chromosomes for a synteny analysis. Through an RNA-seq analysis, 86% (882) PPRs were expressed in flowering buds of upland cotton (G. hirsutum); however, only 11 and 6 were differentially expressed (DE) between restorer R and its near-isogenic (NI) B and between R and its NI A line, respectively. Another RNA-seq analysis identified the expression of only 54% (556) PPRs in 0 and 3 day(s) post-anthesis (DPA) ovules and 24% (247) PPRs in 10 DPA fibers; however, only 59, 6, and 27 PPRs were DE in 0 and 3 DPA ovules, and 10 DPA fibers between two backcross inbred lines (BILs) with differing fiber length, respectively. Only 2 PPRs were DE between Xuzhou 142 and its fiberless and fuzzless mutant. Quantitative RT-PCR analysis confirmed the validity of the RNA-seq results for the gene expression pattern. Therefore, only a very small number of PPRs may be associated with fertility restoration of CMS and genetic differences in fiber initiation and elongation. These results lay a foundation for understanding the roles of PPR genes in cotton, and will be useful in the prioritization of candidate PPR gene functional validation for cotton CMS restoration and fiber development.

Comparative profile analysis reveals differentially expressed microRNAs regulate anther and pollen development in kenaf cytoplasmic male sterility line.

Cytoplasmic male sterility (CMS) is advantageous in extensive crop breeding and represents a perfect model for understanding anther and pollen development research. MicroRNAs (miRNAs) play key roles in regulating various biological processes. However, the miRNA-mediated regulatory network in kenaf CMS occurrence remains largely unknown. In the present study, a comparative deep sequencing approach was used to investigate the miRNAs and their roles in regulating anther and pollen development during CMS occurrence. We identified 283 known and 46 new candidate miRNAs in kenaf anther. A total of 67 differentially expressed miRNAs (DEMs) were discovered between CMS and its maintainer line. Among them, 40 and 27 miRNAs were up- and downregulated, respectively. These 67 DEMs were predicted to target 189 genes. Validation of DEMs and putative target genes were confirmed by using real-time quantitative PCR. In addition, a potential miRNA-mediated regulatory network, which mainly involves the auxin signaling pathway, signal transduction, glycolysis and energy metabolism, gene expression, transmembrane transport, protein modification and metabolism, and floral development, that mediates anther development during CMS occurrence was proposed. Taken together, our findings provide a better understanding of the molecular mechanism of miRNA regulation in pollen development and CMS occurrence in kenaf.

Candidate Gene Selection for Cytoplasmic Male Sterility in Pepper (Capsicum annuum L.) through Whole Mitochondrial Genome Sequencing.

Cytoplasmic male sterility (CMS), which is controlled by mitochondrial genes, is an important trait for commercial hybrid seed production. So far, genes controlling this trait are still not clear in pepper. In this study, complete mitochondrial genomes were sequenced and assembled for the CMS line 138A and its maintainer line 138B. The genome size of 138A is 504,210 bp, which is 8618 bp shorter than that of 138B. Meanwhile, more than 214 and 215 open reading frames longer than 100 amino acids (aas) were identified in 138A and 138B, respectively. Mitochondrial genome structure of 138A was quite different from that of 138B, indicating the existence of recombination and rearrangement events. Based on the mitochondrial genome sequence and structure variations, mitochondrion of 138A and FS4401, a Korean origin CMS line, may have inherited from a common female ancestor, but their CMS traits did originate separately. Candidate gene selection was performed according to the published characteristics of the CMS genes, including the presence SNPs and InDels, located in unique regions, their chimeric structure, co-transcription, and transmembrane domain. A total of 35 ORFs were considered as potential candidate genes and 14 of these were selected, with orf300a and 0rf314a as strong candidates. A new marker, orf300a, was developed which did co-segregate with the CMS trait.

Comparative Transcriptome Analysis Reveals the Cause for Accumulation of Reactive Oxygen Species During Pollen Abortion in Cytoplasmic Male-Sterile Kenaf Line 722HA.

Cytoplasmic male sterility (CMS) is a maternally inherited trait used for hybrid production in plants, a novel kenaf CMS line 722HA was derived from the thermo-sensitive male-sterile mutant 'HMS' by recurrent backcrossing with 722HB. The line 722HA has great potential for hybrid breeding in kenaf. However, the underlying molecular mechanism that controls pollen abortion in 722HA remains unclear, thus limiting the full utilization of this line. To understand the possible mechanism governing pollen abortion in 722HA, cytological, transcriptomic, and biochemical analyses were carried out to compare the CMS line 722HA and its maintainer line 722HB. Cytological observations of the microspore development revealed premature degradation of the tapetum at the mononuclear stage, which resulted in pollen dysfunction. The k-means clustering analysis of differentially expressed genes (DEGs) revealed that these genes are related to processes associated with the accumulation of reactive oxygen species (ROS), including electron transport chain, F1F0-ATPase proton transport, positive regulation of superoxide dismutase (SOD), hydrogen peroxide catabolic, and oxidation-reduction. Biochemical analysis indicated that ROS-scavenging capability was lower in 722HA than in 722HB, resulting in an accumulation of excess ROS, which is consistent with the transcriptome results. Taken together, these results demonstrate that excessive ROS accumulation may affect the normal development of microspores. Our study provides new insight into the molecular mechanism of pollen abortion in 722HA and will promote further studies of kenaf hybrids.

Exploration of miRNAs and target genes of cytoplasmic male sterility line in cotton during flower bud development.

Cytoplasmic male sterility (CMS) lines provide crucial material to harness heterosis for crop plants, which serves as an important strategy for hybrid seed production. However, the molecular mechanism remains obscure. Although microRNAs (miRNAs) play important roles in vegetative growth and reproductive growth, there are few reports on miRNAs regulating the development of male sterility in Upland cotton. In present study, 12 small RNA libraries were constructed and sequenced for two development stages of flower buds from a CMS line and its maintainer line. Based on the results, 256 novel miRNAs were allocated to 141 new miRNA families, and 77 known miRNAs belonging to 54 conserved miRNA families were identified as well. Comparative analysis revealed that 61 novel and 10 conserved miRNAs were differentially expressed. Further transcriptome analysis identified 232 target genes for these miRNAs, which participated in cellular developmental process, cell death, pollen germination, and sexual reproduction. In addition, expression patterns of typical miRNA and the negatively regulated target genes, such as PPR, ARF, AP2, and AFB, were verified by qRT-PCR in cotton flower buds. These targets were previously reported to be related to reproduction development and male sterility, suggesting that miRNAs might act as regulators of CMS occurrence. Some miRNAs displayed specific expression profiles in special developmental stages of CMS line and its fertile hybrid (F1). Present study offers new information on miRNAs and their related target genes in exploiting CMS mechanism, and revealing the miRNA regulatory networks in Upland cotton.

Comparative phosphoproteomic analysis reveals differentially phosphorylated proteins regulate anther and pollen development in kenaf cytoplasmic male sterility line.

Cytoplasmic male sterility (CMS) is widely used in plant breeding and represents a perfect model to understand cyto-nuclear interactions and pollen development research. Protein phosphorylation is ubiquitous and is involved in the regulation of diverse cellular processes. To reveal the possible mechanism of CMS and pollen development in kenaf, we performed an iTRAQ-based comparative phosphoproteome analysis in the anthers of a CMS line and wild-type plant (Wt). Whole transcriptome unigenes of kenaf as the reference genome, we identified a total of 3045 phosphorylated sites on 1640 peptides corresponding to 974 unique proteins. 292 of the peptides which corresponding to 247 unique proteins were differentially phosphorylated (fold change ≥ 1.20 with P value< 0.05) between these two materials. 113 and 134 proteins were characterized as up-regulated or down-regulated phosphorylated, respectively. An evaluation of the phosphoproteome and proteomic results indicated that the most significantly phosphorylated proteins were not associated with abundant changes at the protein level. Bioinformatics analysis demonstrated that many of these proteins were involved in various biological processes which may play key roles in pollen development, including carbohydrate metabolism, energy metabolism, transport, gene expression regulation, signal transduction, and cell cycle control. Our results provide insight into the CMS mechanism and pollen development in kenaf from a protein phosphorylation perspective.

Transcriptome and Hormone Comparison of Three Cytoplasmic Male Sterile Systems in Brassica napus.

The interaction between plant mitochondria and the nucleus markedly influences stress responses and morphological features, including growth and development. An important example of this interaction is cytoplasmic male sterility (CMS), which results in plants producing non-functional pollen. In current research work, we compared the phenotypic differences in floral buds of different Brassica napus CMS (Polima, Ogura, Nsa) lines with their corresponding maintainer lines. By comparing anther developmental stages between CMS and maintainer lines, we identified that in the Nsa CMS line abnormality occurred at the tetrad stage of pollen development. Phytohormone assays demonstrated that IAA content decreased in sterile lines as compared to maintainer lines, while the total hormone content was increased two-fold in the S2 stage compared with the S1 stage. ABA content was higher in the S1 stage and exhibited a two-fold decreasing trend in S2 stage. Sterile lines however, had increased ABA content at both stages compared with the corresponding maintainer lines. Through transcriptome sequencing, we compared differentially expressed unigenes in sterile and maintainer lines at both (S1 and S2) developmental stages. We also explored the co-expressed genes of the three sterile lines in the two stages and classified these genes by gene function. By analyzing transcriptome data and validating by RT-PCR, it was shown that some transcription factors (TFs) and hormone-related genes were weakly or not expressed in the sterile lines. This research work provides preliminary identification of the pollen abortion stage in Nsa CMS line. Our focus on genes specifically expressed in sterile lines may be useful to understand the regulation of CMS.

Comparative Analysis of the Cytology and Transcriptomes of the Cytoplasmic Male Sterility Line H276A and Its Maintainer Line H276B of Cotton (Gossypium barbadense L.).

In this study, the tetrad stage of microspore development in a new cotton (Gossypium barbadense L.) cytoplasmic male sterility (CMS) line, H276A, was identified using paraffin sections at the abortion stage. To explore the molecular mechanism underlying CMS in cotton, a comparative transcriptome analysis between the CMS line H276A and its maintainer line H276B at the tetrad stage was conducted using an Illumina HiSeq 4000 platform. The comparison of H276A with H276B revealed a total of 64,675 genes, which consisted of 59,255 known and 5420 novel genes. An analysis of the two libraries with a given threshold yielded a total of 3603 differentially expressed genes (DEGs), which included 1363 up- and 2240 down-regulated genes. Gene Ontology (GO) annotation showed that 2171 DEGs were distributed into 38 categories, and a Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that 2683 DEGs were classified into 127 groups. Thirteen DEGs were randomly selected and detected by quantitative reverse-transcribed PCR (qRT-PCR), and the results indicated that the transcriptome sequencing results were reliable. The bioinformatic analysis results in conjunction with previously reported data revealed key DEGs that might be associated with the male sterility features of H276A. Our results provide a comprehensive foundation for understanding anther development and will accelerate the study of the molecular mechanisms of CMS in cotton.

A Gene Encoding Pentatricopeptide Repeat Protein Partially Restores Fertility in RT98-Type Cytoplasmic Male-Sterile Rice.

Cytoplasmic male sterility (CMS) lines in rice, which have the cytoplasm of a wild species and the nuclear genome of cultivated rice, are of value for the study of genetic interactions between the mitochondrial and nuclear genomes. The RT98-type CMS line RT98A and the fertility restorer line RT98C carry the cytoplasm of the wild species Oryza rufipogon and the nuclear genome of the Taichung 65 cultivar (Oryza sativa L.). Based on a classical crossing experiment, fertility is reported to be restored gametophytically by the presence of a tentative single gene, designated Rf98, which is derived from the cytoplasm donor. Fine mapping of Rf98 revealed that at least two genes, which are closely positioned, are required for complete fertility restoration in RT98A. Here, we identified seven pentatricopeptide repeat (PPR) genes that are located within a 170 kb region as candidates for Rf98 Complementation tests revealed that the introduction of one of these PPR genes, PPR762, resulted in the partial recovery of fertility with a seed setting rate up to 9.3%. We conclude that PPR762 is an essential fertility restorer gene for RT98-type CMS. The low rate of seed setting suggested that some other genes near the Rf98 locus are also necessary for the full recovery of seed setting.