Skip to main content
  • Research article
  • Open access
  • Published:

Comparative transcriptional analysis of Capsicum flower buds between a sterile flower pool and a restorer flower pool provides insight into the regulation of fertility restoration

Abstract

Background

Cytoplasmic male sterility (CMS) and its restoration of fertility (Rf) system is an important mechanism to produce F1 hybrid seeds. Understanding the interaction that controls restoration at a molecular level will benefit plant breeders. The CMS is caused by the interaction between mitochondrial and nuclear genes, with the CMS phenotype failing to produce functional anthers, pollen, or male gametes. Thus, understanding the complex processes of anther and pollen development is a prerequisite for understanding the CMS system. Currently it is accepted that the Rf gene in the nucleus restores the fertility of CMS, however the Rf gene has not been cloned. In this study, CMS line 8A and the Rf line R1, as well as a sterile pool (SP) of accessions and a restorer pool (RP) of accessions analyzed the differentially expressed genes (DEGs) between CMS and its fertility restorer using the conjunction of RNA sequencing and bulk segregation analysis.

Results

A total of 2274 genes were up-regulated in R1 as compared to 8A, and 1490 genes were up-regulated in RP as compared to SP. There were 891 genes up-regulated in both restorer accessions, R1 and RP, as compared to both sterile accessions, 8A and SP. Through annotation and expression analysis of co-up-regulated expressed genes, eight genes related to fertility restoration were selected. These genes encode putative fructokinase, phosphatidylinositol 4-phosphate 5-kinase, pectate lyase, exopolygalacturonase, pectinesterase, cellulose synthase, fasciclin-like arabinogalactan protein and phosphoinositide phospholipase C. In addition, a phosphatidylinositol signaling system and an inositol phosphate metabolism related to the fertility restorer of CMS were ranked as the most likely pathway for affecting the restoration of fertility in pepper.

Conclusions

Our study revealed that eight genes were related to the restoration of fertility, which provides new insight into understanding the molecular mechanism of fertility restoration of CMS in Capsicum.

Background

Capsicum species are one of the most popular spice and vegetable crops in the world and Capsicum annuum is the most widely grown among the five domesticated species (C. annuum, C. baccatum, C. chinense, C. frutescens, and C. pubescens) [1]. Hybrid vigor is a phenomenon that is advantageous for breeders because of increased fruit yield, enhance resistance, and improve quality. However, the production of F1 hybrid seed needs manual emasculation that can lead to a high cost for F1 seed production [2]. The unique mechanism cytoplasmic male sterility (CMS) is one of the most valuable methods to utilize plant heterosis or hybrid vigor [3] because lack of pollen production removes the need for manual emasculation. Thus, breeders use the CMS/Rf system to produce hybrid seeds more economically, and the system is widely exploited for hybrid seed production of a number of crops including Capsicum [4,5,6].

Male sterility is the failure of plants to produce functional anthers, pollen, or male gametes. The CMS phenotype is maternally inherited because it is controlled by the plant’s mitochondrial genome [4]. It has been proven that CMS is caused by chimeric open reading frames (ORF) resulting from the rearrangements of the mitochondrial genome [7, 8]. These ORF may disturb the function of ATPase [9], destroy the mitochondrial membrane structure [10], and produce proteins that are cytotoxic [11], which, in turn, affect the normal development of pollen [12]. In pepper, CMS was first reported in 1958 from an Indian Capsicum annuum accession (PI164835) [13]. Since then, the CMS/Rf system has been used to produce F1 hybrid seeds of pepper [14]. Two candidate CMS genes, orf456 and atp6-2 loci, have since been identified [15, 16]. Another ORF, designated orf507, is a modified version of orf456, elongated through the deterioration of a stop codon. This ORF was also proven to be related to CMS and inhibit the formation of microspore in pepper [17].

The CMS phenotype can be reversed by a nuclear Rf gene. These genes have been found to restore fertility through several different mechanisms. The CMS transcript can be processed by a Rf gene at the post-transcriptional level [18]. In rice, the transcript of B-atp6-orf79 is silenced by cleavage from the restorer gene RF1A [19]. Restorer of fertility genes can also act at the translational level, as Rfo in radish is thought to repress the translational processing of orf138 [20]. In maize, a putative aldehyde dehydrogenase (rf2) was found that may act to restore fertility through detoxification during pollen development [21].

Restorer-of-fertility genes have been found in a range of flowering plant species. They have been reported in petunia [22], radish [20, 23,24,25,26], sugar beet [27, 28], maize [21], sorghum [29], and rice [19, 30,31,32]. These Rf genes have encoded a variety of different proteins. For example, Rf1 (bvORF20) encodes an OMA1-like protein in sugar beet [27, 28], Rf2 encodes an aldehyde dehydrogenase protein in maize [21], Rf17 gene encodes an acyl-carrier protein synthase [33] and the Rf2 gene encodes a glycine-rich protein in rice [34], indicating the existence of a diverse set of Rf genes. However, most Rf genes are known to encode Pentatricopeptide Repeat (PPR) proteins [8, 35, 36]. In pepper, so far all predicted Rf genes have encoded PPRs [36, 37]. A PPR gene, CaPPR6, was identified as a strong Rf candidate based on expression pattern and characteristics of coding sequence [37]. Recently, 12 candidate PPR genes with similarity to previously reported Rf genes were also identified in pepper [38]. These candidate Rf genes provide a basis for further study of fertility restoration in pepper.

It has been suggested that fertility restorer in pepper may be controlled by two complementary genes [39]. Previous studies show that the fertility restorer of CMS in pepper is controlled by two major additive-dominant epistatic genes and an additive-dominant polygene [40], and two major QTLs and several minor QTLs [41]. It could be that the two major QTLs correspond with the two major additive-dominant genes, in which case the studies support one another. In contrast, another study indicates that one major QTL and four minor QTLs relate to fertility restoration in pepper [42]. Another phenotype of partial restoration has also been reported, in which the flower simultaneously produces functional and aborted pollen, which is thought to be controlled by a gene (pr) in the nucleus separate from Rf genes [43]. In addition, the CMS phenotype can be restored temporarily under low temperature, suggesting that temperature affects the expression of some fertility modification genes [44]. Together, these various types of fertility restoration demonstrate that CMS is complex, and currently do not have a complete understanding of the molecular mechanisms that underlie the CMS/Rf system in pepper.

The RNA-Seq method directly sequences transcripts by using high-throughput sequencing technologies, and it has considerable advantages for providing genome-wide information, detection of novel transcripts, and allele-specific expression [45]. Bulked segregate analysis (BSA) is an efficient method for the rapid identification of molecular markers for specific traits or target gene loci [46]. Combing the advantage of BSA and RNA-seq, BSA RNA-seq (BSR) can be used to analyze the differentially expressed genes (DEGs), and single nucleotide polymorphisms (SNPs) between the two genetic pools [47, 48].

In this study, BSR-seq was applied to identify DEGs related to the fertility restorer of CMS in pepper. In addition, the transcriptomes of two parent lines were sequenced. A set of candidate genes were selected that are associated with the fertility restoration in CMS in pepper based on both the BSR-seq and parental transcriptome sequencing. The results provide new insights into the study of molecular mechanisms of restorer fertility of CMS in pepper.

Results

Database estimation of transcript sequencing

Through the RNA-sequence of the fertility restorer line (R1), the CMS line (8A), a population of 30 fertile plants pool (RP), and a population of 30 sterile plants pool (SP), a total of 41.84 GB of aligned data were obtained. The aligned data of R1 and 8A were 8.24 GB and 6.98 GB, respectively, and that of RP and SP were 14.16 GB and 12.46 GB, respectively (Table 1). These raw data can be found in NCBI (https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/) with an accession number of SRA895207. The base ratios for quality scores of each aligned data was greater than Q30% at more than 91%, and GC content was more than 41%. This indicates that the aligned data were good for the subsequent searches.

Table 1 The estimation of RNA sequence data

The aligned data had a high success rate of being mapped to a reference genome. The percentage of each aligned data being mapped to a reference genome was more than 85.50%, and the unique mapped rate was greater than 82.47% (Table 2).

Table 2 The comparison between sequence data and reference genomics

Differentially Expressed Genes (DEGs) analysis

DEGs were discovered between R1/8A and between RP/SP (Table 3). There were 3790 DEGs between R1 and 8A, in which 2274 were up-regulated and 1516 were down-regulated for expression in R1 as compared to 8A. There were also 1762 DEGs between RP and SP, in which 1490 were up-regulated and 272 were down-regulated for expression in RP as compared to SP. In an overall comparison, there were 944 co-DEGs among the above groups (R1/8A, RP/SP). Within this comparison, 891 out of 944 DEGs were commonly up-regulated in restorer accessions and the remaining 53 out of 944 DEGs were down-regulated in restorer accessions (R1 and RP) as compared to CMS accessions (8A and SP). This indicates that 891 DEGs were up-regulated not only in RP as compared to SP, but also in R1 as compared to 8A, and 53 DEGs were down-regulated not only in RP as compared to SP, but also in R1 as compared to 8A. The 891 commonly up-regulated DEGs were the subject of subsequent study.

Table 3 Three groups of DEGs

Gene Ontology (GO) Annotation

The 891 common up-regulated DEGs in restorer accessions were assigned to three main categories, cellular component, molecular function, and biological process. These three categories were composed of 53 functional groups using GO assignment (Fig.1).

Fig. 1
figure 1

The GO functional classification of up-regulated genes. Red bars represent all expressed genes, and blue bars represent DEGs

In the cellular component category, the majority functional groups were cell part, cell, organelle, and membrane, including 589, 573, 475, and 397 genes, respectively. The most significant GO node that is enriched to the DEGs is external side of plasma membrane (GO:0009897), composed of Capana01g001287, Capana01g00392, Capana06g000150, Capana01g004065 and Capana09g002397 totaling 5 up-regulated DEGs (Table 4). The next were GO:0009535, GO:0005615, GO:0030173, GO:0005618, GO:0048046 and GO:0016021, including 4, 5, 6, 91, 60 and 97 up-regulated DEGs, respectively (Table 4).

Table 4 The enrichment results for the cellular component DEGs by topGO

In the molecular function category, the dominant function types were catalytic activity and binding with 369 and 360 genes, respectively. The most significant GO node that is enriched to the DEGs is serine-type endopeptidase activity (GO:0004252), composed of Capana04g000159, Capana06g000150, Capana09g002397, Capana05g000136, Capana11g002184 and Capana01g001287 totaling 6 up-regulated DEGs (Table 5). The next GO nodes were protein kinase activity (GO:0004672), transmembrane receptor protein kinase activity (GO:0019199), 8-hydroxyquercitin 8-O-methyltransferase activity (GO:0030761) and isoflavone 4'-O-methyltransferase activity (GO:0030746), including 56, 4, 1 and 1 up-regulated DEGs, respectively (Table 5).

Table 5 The enrichment results of molecular function DEGs by topGO

In the biological process category, the dominant terms were metabolic process, response to stimulus, biological regulation, developmental process and cellular component organization or biogenesis including 574, 564, 511, 460, 368, 351, and 332 genes, respectively. The most significant GO node that is enriched to the DEGs is mucilage extrusion from seed coat (GO:0080001), composed of Capana04g000159, Capana05g000136, apana09g002397, Capana01g001287 and Capana06g000150 totaling five up-regulated DEGs (Table 6). The next nodes were GO:0016045, GO:0006898, GO:0010204, GO:0048359, GO:0010359, GO:0010102 and GO:0052544, including 9, 6, 1, 10, 7, 9 and 8 up-regulated DEGs, respectively (Table 6).

Table 6 The enrichment results of the biological process DEGs by topGO

Kyoto Encyclopedia of Genes and Genomes (KEGG ) Metabolic Pathway of DEGs

The up-regulated shared DEGs were annotated to 49 KEGG metabolic pathways in five categories including genetic information processing, metabolism, organismal systems, cellular processes, and environmental information processing (Fig. 2). The metabolic pathways composed of the most up-regulated DEGs were starch and sucrose metabolism, oxidative phosphorylation, and plant-pathogen interaction. The next metabolic pathways composed of nine up-regulated DEGs were inositol phosphate metabolism, and the phosphatidylinositol signaling system (Fig. 2). KEGG pathways that may be involved in fertility recovery or pollen development include energy metabolism, carbohydrate metabolism, protein and amino acid metabolism, lipid metabolism, substance absorption and transport, and signal transduction (Additional File 1: Table S1). Interestingly, two metabolic pathways, phosphatidylinositol signaling system and inositol phosphate metabolism, were enriched as the most reliable pathway for enrichment significance (Fig. 3). Within these two metabolic pathways most genes were the same, with eight out of nine genes being shared between the two pathways.

Fig. 2
figure 2

Statistic analysis of up-regulated differentially expressed genes in KEGG pathways

Fig. 3
figure 3

Scatter plot of KEGG pathway enrichment for up-regulated DEGs. Each small shape in the figure represents a KEGG path, and the path names are shown in the legend on the right. The horizontal coordinate is the enrichment factor, the smaller the enrichment factor, the more significant the enrichment level of DEGs in the pathway. The ordinate is -log10 (Q value), the larger the ordinate, the more reliable the enrichment of DEGs in the pathway

In the phosphatidylinositol signaling system (ko04070), scored as the most enriched system, nine genes were up-regulated (Fig. 4). Among these nine genes, five (Capana00g002844; Capana00g004424; Capana10g001436; Capana10g002170; Capana10g002470) encode phosphatidylinositol-4-phosphate 5-kinase (PI(4)P5K) and catalyzes the phosphatidylinositol-4-phosphate (PI(4)P) to phosphatidylinositol-4,5-biphosphate (PI(4,5)P2). Meanwhile, two genes (Capana03g002795 and Capana06g002131) encode phospholipase C (PLC), which could hydrolyze phosphatidylinositol (PI), PI(4)P or PI(4,5)P2 to generate double messenger molecules inositol triphosphate (IP3) and diacylglycerol (DG). Two genes (Capana05g000173 and Capana07g002321) encode inositol phosphate phosphatase and phosphatidate cytidylyltransferase, respectively, which could participate in the reduction of IP3 and DG to PI.

Fig. 4
figure 4

The gene node participating in phosphatidylinositol signaling system. The red square frame means up-regulated expressed genes in the node

Related Genes Selection and expression

According to conjoined analysis of the gene FPKM (fragments per kilobase of exon per million fragments mapped) value, difference multiple, qRT-PCR value, functional annotation and metabolic pathway classification, eight genes, related to fertility restoration of CMS in Capsicum were selected. The eight genes were Capana00g002348, Capana00g004424, Capana02g000930, Capana00g003267, Capana01g002849, Capana05g002270, Capana01g004065 and Capana06g002131, and they encode fructose kinase, phosphatidylinositol phosphokinase, pectin lyase, extragalacturonase, pectin esterase, cellulose synthase, and bundle arabinogalactan protein, respectively (Table 7). All listed genes are key enzymes and proteins in anther and pollen development.

Table 7 The selected genes related to the restorer-of-fertility and their expression and function annotation

According to RNA-sequencing, these genes were dramatically up-regulated in the restorer parent (R1) and restorer pool (RP). The majority of these selected genes related to fertility restoration showed very little expression in the CMS 8A and SP plants, and one selected gene, Capana00g004424, had no detected expression in CMS line 8A and SP (Table 7). The lack of expression of these genes was validated by qRT-PCR between 8A and R1, as well as SP and RP. The qRT-PCR results indicated that these genes were up-regulated in both R1 and RP, as compared to 8A and SP, which was completely consistent with the sequencing results (Fig. 5).

Fig. 5
figure 5

The qRT-PCR validation of eight genes related to fertility restorer between 8A and R1, as well as SP and RP. (a) The relative expression of Capana00g002348 in four accessions. (b) The relative expression of Capana00g004424 in four accessions. (c) The relative expression of Capana02g000930 in four accessions. (d) The relative expression of Capana00g003267 in four accessions. (e) The relative expression of Capana01g002849 in four accessions. (f) The relative expression of Capana05g002270 in four accessions. (g) The relative expression of Capana01g004065 in four accessions. (h) The relative expression of Capana06g002131 in four accessions

The relative expression of two obviously down-regulated genes (NewGene11661 and NewGene949) both in fertile accessions R1 and RP as compared to sterile accessions 8A and SP were validated by qRT-PCR. The results indicated that two genes were also down-regulated in R1 compared to 8A (Fig. 6).

Fig. 6
figure 6

The qRT-PCR validation of two obviously down-regulated genes between 8A and R1

Tissue from four different developmental stage buds tested the relative expression, and the different stages are shown in Fig. 7. The qRT-PCR results show that the expression of these genes had a lower and relatively stable level among four developmental stages of flower buds in 8A. However, in the F1 generation, the relative expression varied among the different developmental stages. In the F1 generation, as the flower buds developed, the relative expression had a dramatic increase. At stage III, the expression increased rapidly until it peaked at stage IV (Fig. 8).

Fig. 7
figure 7

The four developmental stages of flower buds used for qRT-PCR analysis

Fig. 8
figure 8

The expression of genes related to fertility restorer in different developmental stage of buds between 8A and F1. (a) The relative expression of Capana00g002348. (b) The relative expression of Capana00g004424. (c) The relative expression of Capana02g000930. (d) The relative expression of Capana00g003267 in four accessions. (e) The relative expression of Capana01g002849. (f) The relative expression of Capana05g002270. (g) The relative expression of Capana01g004065. (h) The relative expression of Capana06g002131

In addition, the relative expression of two down-regulated genes were analyzed in four developmental stages of the buds between 8A and F1. The relative expression of NewGene11661 improved with the development of flower buds in two accessions, and the expression in every stage of 8A were higher than in R1 (Fig. 9). Unfortunately, although the relative expression of NewGene949 was higher in every stage in 8A than in R1, there was not agreement with the expected tendency (Fig. 9).

Fig. 9
figure 9

The relative expression of two down-regulated genes in different developmental stage of buds between 8A and F1. (a) The relative expression of NewGene11661. (b) The relative expression of NewGene949

Discussion

Male sterility and the fertility restorer system are an extremely complex biological process involving substance and energy metabolism, signal transduction pathway, substance transportation, pollen wall morphogenesis, tapetum formation and programmed cell death (PCD), and a series of related gene expression regulation processes.

Anthers are the strongest energy reservoir in the flower organs, and have a very active metabolism during development. A large number of sugars are transported to the anther [49], which can regulate the expression of genes as both a substrate of carbohydrate and a signal molecule in anthers [50]. In this study, one gene encoding fructokinase had an up-regulated expressed value of log2FC more than 8.5 times in R1/8A as well as RP/SP in a sucrose and starch metabolism pathway, which indicated that fructokinase is one of the key genes that regulates the fertility restorer of CMS in pepper.

Generally, pectin and callose in pollen mother cell (PMC) are degraded absolutely, otherwise the microspore cannot be separated from the tetrad [51, 52]. The pectin methylesterification, is degraded by de-methylated esterification first by methylesterases (PMEs), then the combined action of PME, pectin lyases (PLs), and polygalacturonases (PGs) [53, 54]. In Arabidopsis, QRT1 gene encodes a PME, QRR2, and QRT3 encoding an external PG and an endonuclear PG, respectively. The loss of function of QRT1 didn’t reduce the level of pectin methylesterification. If the level of the pectin methylesterification is higher, it cannot be degraded by PG [55]. In this study, three genes encoding PME, PL and exo-PG were selected. What is more, these genes had up-regulated expression and the log2FC were as high as 11.4473, 10.2657, and 8.97438, respectively. The results presented are in agreement with Hamid et al [56] that showed that eight pectin lyase-like superfamily protein coding genes and five pectin methylesterase genes were up-regulated in fertile plants as compared to Cytoplasmic Genic Male Sterility (CGMS) in cotton. In addition, a fasciclin-like protein gene was also up-regulated in restorer materials in this study. This result was similar to the reports of Hamid et al. [56], in which three cytoskeleton organization genes were up-regulated in fertile lines in cotton. These up-regulated genes may have a positive role in the degradation of pectin and the normal release of microspores.

It is well known that celluloses and hemicelluloses are the important components of pollen intine. Cellulose composed of a class of β-1,4-glucan molecular long chain plays an important role in cell wall toughness and strength. The deposition of cellulose on intine is mainly carried out by cellulose synthase complexes (CSCs) that is located on the cell membrane [57, 58]. In Arabidopsis, the deposition of cellulose in intine and extine was not well-distributed in the mutant of CESA1 gene and CESA3 gene, which resulted in abnormal pollen development. Fortunately, we also choose one gene encoding cellulose synthase that is up-regulated dramatically in restorer accessions.

It is commonly thought that the phosphatidylinositol signal system is one of the important signal systems for plant seed germination, growth and reproduction, senescence and response to environmental factors [59]. Pollen development undergoes a series of complex cell division and differentiation processes, which involve the dynamic changes of many cell components and internal subcellular components, including vacuole and cytoskeleton [60]. Many components of phosphatidylinositol signaling system participate in the vacuolar diversification during pollen development and vesicle transport in pollen tube growth. In this study, eight genes were up-regulated in phosphatidylinositol signal system. There are five genes that encode PI(4)P5K, which can catalyze PI(4)P to PI(4,5)P2. Previous studies have shown that PI(4)P is very important for pollen and stigma affinity, and PI(4,5)P2 plays an important role in vesicle transport and cell skeleton rearrangement [61]. PI(4)P5K is a very important enzyme in the development of the anther, it would lead to an abnormal morphology of the pollen tube if PI(4)P5K was lost, thus inhibiting the germination of pollen and the growth of the pollen tube [62,63,64,65]. In Arabidopsis, PIP5K1 and PIP5K2 are important for vacuole biogenesis and early pollen development, pollen grains from flowers of the pip5k1+/−pip5k2+/−mutants show defects in vacuoles and exine wall formation [65]. In addition, two genes encode PLC, and PLC is the most important in phosphatidylinositol signal system, which could hydrolyze PI, PI(4)P and PI(4,5)P2, to double as the messenger molecules inositol trisphosphate (IP3) and diacylglycerol (DG). It is also known that there is a calcium dependent PLC activity in pollen tubes, and PLC and IP3 are involved in the germination and growth of pollen tubes [66]. PLC can monitor the growth of the pollen tube not only by adjusting the content of PI(4,5)P2, but also by regulating the concentration of Ca2+ and membrane secretion.

The development of pollen walls plays a key role in the development of pollen, and it leads to male sterility in plants if the development of the pollen outer wall is blocked. Sporopollenin is the main substance that constitutes the outer wall of pollen, and the sporopollenins are mainly composed of polymerized phenols and long-chain fatty acid derivatives, which are difficult to decompose and can effectively protect pollen from degradation from the outside. There is also a thick oil-bearing layer outside of the pollen grain, including hydrophobic lipids and proteins, composed of long-chain fatty acid derivatives [67, 68]. Previous studies showed that sporopollenin biosynthesis is closely associated with fatty acid metabolism [69, 70]. Therefore, lipid accumulation plays an important role in pollen morphogenesis. In this study, some up-regulated genes related to lipid metabolism pathway were discovered, and these metabolic genes may be involved in the synthesis of lipids in the tapetum, which are then transported to the pollen wall surface (Additional file 1: Table S1). It had also been reported in cabbage that in the protein interaction network around CYP704B1, more than 1/3 of the protein species were involved in fatty acid metabolism, and all of them were significantly reduced in 83121A [71].

Vesicle transport and lipid carrier transport are the two main modes of sporopollen transportation. Adenosine triphosphate binding cassette (ABC) is a protein family in which ABC members were involved in the transmembrane transport of substances. In Arabidopsis thaliana, more than 120 ABC proteins have been identified. They are involved in the transport of ions, hormones, secondary metabolites, lipids, and proteins. The ABCG26/GBC27 belongs to the G subfamily of ABC protein and is expressed in microspore and tapetum, which may be involved in the transport of sporopollen [72, 73]. In this study, two up-regulated ABC transport genes were discovered that may be involved in the transportation of sporopollen (Additional file 1: Table S1).

In addition, the synthesis of fatty acid needs ATP and acetyl-CoA that come from cell respiration and energy metabolism. Plant energy metabolism mainly includes the tricarboxylic acid cycle, glycolysis, pentose phosphate pathway, and the electron transport and oxidative phosphorylation. This energy metabolism provides ATP and acetyl-CoA for the synthesis of fatty acids. Previous studies have shown that the impairment of respiratory chain enzymes and enzyme complexes can lead to CMS [74, 75]. Similar to the results in eggplant [76], this study found many up-regulated genes in the restorer line to participate in energy pathways, such as oxidative phosphorylation, glycolysis, and citrate cycle (Additional file 1: Table S1).

The relative expression of NewGene11661 was down-regulated in the F1 while up-regulated in 8A, and the expression increased with the development of flower buds. However, it did not improve more dramatically than other candidate genes. NewGene11661 was annotated as UDP-glycosyltransferase gene. The UDP glycosyltransferase could catalyze conjugation of lipophilic chemicals with sugar donated by UDP-glycoside, generating water-soluble products that can be easily excreted and resulting in the detoxification and elimination of their substrate [77, 78]. However, the roles of this enzyme in the development pollen or CMS have been rarely reported and needs further study.

Conclusion

By RNA sequencing, in this study 891 DEGs were up-regulated and 53 DEGs were down-regulated in restorer accessions as compared to the CMS accessions. The 891 up-regulated DEGs in restorer accessions were assigned to three main functional categories: cellular component, molecular function, and biological process, all of which were composed of 53 functional groups using GO assignment. The up-regulated DEGs were annotated to 49 KEGG metabolic pathways in five categories including genetic information processing, metabolism, organismal systems, cellular processes, and environmental information processing. As in Fig. 3, two metabolic pathways, phosphatidylinositol signaling system and inositol phosphate metabolism, were statistically the most likely pathways for affecting fertility restoration. Finally, eight DEGs were selected that encoded fructose kinase, phosphatidylinositol phosphokinase, pectin lyase, extragalacturonase, pectin esterase, cellulose synthase, and bundle arabinogalactan protein. The qRT-PCR results showed that the expression of these genes had a lower and relatively stable level between four developmental stages of the flower bud in 8A. But in the F1 generation, with the development of flower buds, the relative expression had a tendency to significant increase, especially at the stage III, where the expression increased rapidly and peaked at stage IV. The tendency to significantly increase the eight candidate genes supports RNA differential expression and are likely to be involved in pepper fertility restoration.

Materials and Methods

Plant sample collection and preparation

The CMS 8A line was completely sterile, and the fertility restorer line R1 completely restored the fertility of 8A (Fig. 10). The F2 population from the self-pollination of the F1 individuals showed segregation for fertility. The restore fertility pool (RP) was constructed from 30 polar fertile plants and the sterile pool (SP) was constructed from 30 polar sterile plants. The flower buds of 8A, R1, SP, and RP at the tetrad stage were used for total RNA extraction. Four different developmental stages of flower buds of 8A and F1 analyzed the relative expression of fertility restoration related genes.

Fig. 10
figure 10

The flower pictures of CMS line 8A and fertility restorer line R1

RNA extraction and library construction and RNA Sequencing

Total RNA was extracted separately from the flower buds of 8A, R1, SP, and RP according to the instructions of Trizol Reagent (Life technologies, California, USA). The RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). The RNA concentration was measured using Qubit® RNA Assay Kit in Qubit®2.0 Flurometer (Life Technologies, CA, USA). The RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Then the mRNA was enriched using magnetic beads with Oligo (dT) for reverse transcription. The cDNA library was constructed following the manufacturer’s instructions for NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, E7530). Finally, the cDNA libraries of the four flower buds from 8A, R1, SP, and RP have been sequenced on a flow cell using the Illumina HiSeq™ 2500 sequencing platform.

Transcriptome analysis using reference genome-based reads mapping

Low quality reads, such as adaptor sequences, unknown nucleotides greater than 5%, or low quality score (Q-value)≤ 20 bases, were removed by Perl script. The aligned reads that were filtered from the raw reads were mapped to the Pepper Genome Database (release 2.0) [79] using TopHat2 software [80]. The aligned records in BAM/SAM format examined to remove potential duplicate molecules. Gene expression levels were estimated using FPKM values by the Cufflinks software [81]. Identification of new genes was based on transcript discovery with Cufflinks, and then filtering the genes encoding for a short peptide chain (less than 50 amino acid residues). By comparing the raw gene model and the result of transcriptome assembly, gene structure was optimized. SpliceGrapher integrated splice graphs generated from Cufflinks output and read alignments to produce comprehensive alternative splicing predictions [82].

Identification of DEGs

To evaluate DEGs among lines 8A, R1, SP, and RP, the EBSeq [83] was employed with screening criteria FC ≥ 2 / log2FC ≥ 1 and False Discovery Rate (FDR) < 0.01. The differential multiple / FC represents the ratio of the expression between the two groups. By adjusting the difference between P-values, the FDR is obtained. Because the differential expression analysis of transcriptome sequencing is an independent statistical hypothesis test for a large number of gene expression values, there is a false positive problem. To address this, in the process of differential expression analysis, the accepted Benjamini-Hochberg correction method corrected the significant P-value obtained from the original hypothesis test, and finally FDR was used as the key index for screening DEGs.

GO classification and enrichment analysis

Perl script plotted GO functional classification for the unigenes with a GO term hit to view the distribution of gene functions. GO enrichment analysis of the DEGs was implemented by the GOseq R packages based Wallenius non-central hyper-geometric distribution [84], which can adjust for gene length bias in DEGs.

KEGG pathway and enrichment analysis

KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism, and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies (http://www.genome.jp/kegg/) [85]. This study used KOBAS software to test the statistical enrichment of differential expression genes in KEGG pathways [86].

Screening of fertility restoration related genes

Genes related to fertility were selected according to comprehensive estimation of gene log2FC value and gene function. Firstly, genes that were dramatically up-regulated (log2FC ≥ 7) in restorer accessions as compared to sterile accessions were selected. Genes were selected on the basis of having FPKM values higher in restorer accessions and having FPKM values close to zero in sterile accessions (Table 7). Secondly, this group was filtered according to function, choosing genes playing an important role in the normal development of pollen as found in previous studies, or are involved mostly in the important or enriching pathways in this study to further evaluate.

Expression analysis of fertility restoration related genes

Extraction of RNA was performed under the conditions described above. The first strand cDNA synthesis was carried out using the 1st strand cDNA synthesis kit (Revert Aid Premium Reverse Transcriptase) (Thermo Scientific, EP0733). A total of eight fertility restorer related genes and two down-regulated genes were tested for their relative expression and the CaActin (GenBank Accession: GQ339766.1) gene was the internal control (Table 8). The total volume of each reaction is 20 μL. Using SG Fast qPCR Master Mix (High Rox) (2×), qRT-PCR was carried out (BBI, B639273). The qRT-PCR was performed on an ABI Stepone plus Real-Time PCR System (Applied Biosystems, USA) with the following cycling parameters: 95°C for 3 m, followed by 45 cycles of: 95 °C for 7 s, 57°C for 10 s, 72°C for 15 s. The relative expression was calculated by using the 2−ΔΔCt method described by Livak and Schmittgen [87]. All reactions were performed with at least three replicates.

Table 8 The test genes and their primers for qRT-PCR

Availability of data and materials

All data generated and analyses done within this study are included in this published article and its supplementary information files. The raw data can be found in NCBI (https://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/) with an accession number of SRA895207.

Abbreviations

ABC:

triphosphate binding cassette

BSA:

Bulked segregate analysis

BSR:

BSA RNA-seq

CGMS:

Cytoplasmic Genic Male Sterility

CMS:

Cytoplasmic male sterility

CSCs:

Cellulose synthase complex

DEG:

Differentially expressed gene

DG:

Diacylglycerol

FC:

Fold Change

FDR:

Discovery Rate

FPKM:

Fragments per kilobase of exon per million fragments mapped

GO:

Gene Ontology

IP3 :

Inositol triphosphate

KEGG:

Kyoto Encyclopedia of Genes and Genomes

ORF:

Open reading frames

PCD:

Programmed cell death

PG:

Polygalacturonase

PI (4) P:

Phosphatidylinositol-4-phosphate

PI (4) P5K:

Phosphatidylinositol-4-phosphate 5-kinase

PI (4,5) P2 :

phosphatidylinositol-4,5-biphosphate

PI:

Phosphatidylinositol

PL:

Pectin lyase

PLC:

Phospholipase C

PMC:

Pollen mother cell

PME:

Methylesterase

PPR:

Pentatricopeptide Repeat

qRT-PCR:

quantitative real-time polymerase chain reaction

QTL:

Quantitative trait loci

Rf:

Fertile restorer / restoration of fertility

RP:

Restorer pool

SP:

Sterile pool

References

  1. Bosland PW, Votava EJ. Peppers. In: Vegetable and spice capsicums. 2nd ed. Wallingford: CAB International; 2012. p. 230.

  2. Colombo N, Galmarini CR. The use of genetic, manual and chemical methods to control pollination in vegetable hybrid seed production: a review. Plant Breeding. 2017;136:287–99.

    Article  Google Scholar 

  3. Kaul MLH. Male Sterility in Higher Plants. Berlin, Heidelberg: Springer-Verlag; 1988.

    Book  Google Scholar 

  4. Ji JJ, Huang W, Yin YX, Li Z, Gong ZH. Development of a SCAR marker for early identification of S-cytoplasm based on mitochondrial SRAP analysis in pepper (Capsicum annuum L.). Mol Breeding. 2014;33:679–90.

    Article  CAS  Google Scholar 

  5. Havey MJ. The Use of Cytoplasmic Male Sterility for Hybrid Seed Production. In: Daniell H, Chase C, editors. Molecular Biology and Biotechnology of Plant Organelles. Dordrecht: Springer; 2004. p. 623–34.

    Chapter  Google Scholar 

  6. Kumar S, Singh V, Singh M, Rai S, Kumar S, Rai SK, Rai M. Genetics and distribution of fertility restoration associated RAPD markers in pepper (Capsicum annuum L.). Sci Hortic. 2007;111:197–202.

  7. Hanson MR, Bentolila S. Interactions of mitochondrial and nuclear genes that affect male gametophyte development. The Plant Cell, Supplement. 2004;16:S154–S69.

    Article  CAS  Google Scholar 

  8. Chase CD. Cytoplasmic male sterility: A window to the world of plant mitochondrial-nuclear interactions. Trends Genet. 2007;23:81–90.

    Article  CAS  PubMed  Google Scholar 

  9. Dieterich JH, Braun HP, Schmitz UK. Alloplasmic male sterility in Brassica napus (CMS ‘Tournefortii-Stiewe’) is associated with a special gene arrangement around a novel atp9 gene. Mol Genet Genomics. 2003;269:723–31.

    Article  CAS  PubMed  Google Scholar 

  10. Jing B, Heng S, Tong D, Wan Z, Fu T, Tu J, Ma CZ, Yi B, Wen J, Shen JX. A male sterility-associated cytotoxic protein ORF288 in Brassica juncea causes aborted pollen development. J Exp Bot. 2012;63:1285–95.

    Article  CAS  PubMed  Google Scholar 

  11. Peng XJ, Li FH, Li SQ. Expression of a mitochondrial gene orfH 79 from the CMS-HongLian rice inhibits Saccharomyces cerevisiae growth and causes excessive ROS accumulation and decrease in ATP. Biotechnol Lett. 2009;31:409–14.

    Article  CAS  PubMed  Google Scholar 

  12. Tuteja R, Saxena RK, Davila J, Shah T, Chen W, Xiao YL, Fan G, Saxena KB, Alverson AJ, Spillane C, Town C, Varshney RK. Cytoplasmic male sterility-associated chimeric open reading frames identified by mitochondrial genome sequencing of four cajanus genotypes. DNA Res. 2013;20:485–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Peterson PA. Cytoplasmically inherited male sterility in Capsicum. Amer Natl. 1958;92:111–9.

    Article  Google Scholar 

  14. Swamy BN, Hedau NK, Chaudhari GV, Kant L, Pattanayak A. CMS system and its stimulation in hybrid seed production of Capsicum annuum L. Sci Hortic. 2017;222:175–9.

    Article  Google Scholar 

  15. Kim DH, Kang JG, Kim BD. Isolation and characterization of cytoplasmic male sterility-associated orf456 gene of chili pepper (Capsicum annuum L.). Plant Mol Biol. 2007;63:519–32.

    Article  CAS  PubMed  Google Scholar 

  16. Kim DS, Kim BD. The organization of mitochondrial atp6 gene region in male fertile and CMS lines of pepper (Capsicum annuum L.). Curr Genet. 2006;49:59–67.

    Article  CAS  PubMed  Google Scholar 

  17. Gulyas G , Shin Y , Kim H , Lee JS, Hirata Y. Altered transcript reveals an orf507 sterility-related gene in chili pepper (Capsicum annuum L.). Plant Mol Biol Rpt. 2010;28:605–12.

    Article  CAS  Google Scholar 

  18. Chen LT, Liu YG. Male sterility and fertility restoration in crops. Annu Rev Plant Biol. 2014;65:579–606.

    Article  CAS  PubMed  Google Scholar 

  19. Wang Z, Zou Y, Li X, Zhang Q, Chen L, Wu H, Su D, Chen Y, Guo J, Luo D, Long Y, Zhong Y, Liu Y. Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing. Plant Cell. 2006;18:676–87.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Uyttewaal M, Arnal N, Quadrado M, Martin-Canadell A, Vrielynck N, Hiard S, Gherbi H, Bendahmane A, Budar F, Mireau H. Characterization of Raphanus sativus pentatricopeptide repeat proteins encoded by a fertility restorer locus for Ogura cytoplasmic male sterility. Plant Cell. 2008;20:3331–45.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Cui X, Wise RP, Schnable PS. The rf2 nuclear restorer gene of male-sterile T-cytoplasm maize. Sci. 1996;272:1334–6.

    Article  CAS  Google Scholar 

  22. Bentolila S, Alfonso AA, Hanson MR. A pentatricopeptide repeat-containing gene restores fertility to cytoplasmic male sterile plants. Proc Natl Acad Sci USA. 2002;99:10887–192.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Brown GG, Formanova N, Jin H, Wargachuk R, Dendy C, Patil P, Laforest M, Zhang J, Cheung WY, Landry BS. The radish Rfo restorer gene of Ogura cytoplasmic male sterility encodes a protein with multiple pentatricopeptide repeats. Plant J. 2003;35:262–72.

    Article  CAS  PubMed  Google Scholar 

  24. Deslorie S, Gherbi H, Laloui W, Maradour S, Clouet V, Cattolico L, Falentin C, Giancola S, Renard M, Budar F, Small ID, Caboche M, Delourme R, Bendahmane A. Identification of the fertility restoration locus, Rfo, in radish, as a member of the pentatricopeptide-repeat protein family. EMBO Rpt. 2003;4:588–94.

    Article  CAS  Google Scholar 

  25. Koizuka N, Imai R, Fujimoto H, Hayakawa T, Kimura Y, Kohno-Murase J, Sakai T, Kawasaki S, Imamura J. Genetic characterization of a pentatricopeptide repeat protein gene, orf687, that restores fertility in the cytoplasmic male-sterile Kosena radish. Plant J. 2003;34:407–15.

    Article  CAS  PubMed  Google Scholar 

  26. Wang Z, Wang C, Mei S, Gao L, Zhou Y, Wang T. An insertion-deletion at a pentatricopeptide repeat locus linked to fertility transition to cytoplasmic male sterility in radish (Raphanus sativus L.). Mol Breeding. 2015;25:1–5.

  27. Matsuhira H, Kagami H, Kurata M, Kitazaki K, Matsunaga M, Hamaguchi Y, Hagihara E, Ueda M, Harada M, Muramatsu A, Yui-Kurino R, Taguchi K, Tamagake H, Mikami T, Kubo T. Unusual and typical features of a novel restorer-of-fertility gene in sugar beet (Beta vulgaris L.). Genet. 2012;192:1347–58.

    Article  CAS  Google Scholar 

  28. Kitazaki K, Arakawa T, Matsunaga M, Yui-Kurino R, Matsuhira H, Mikami T, Kubo T. Post-translational mechanisms are associated with fertility restoration of cytoplasmic male sterility in sugar beet. Plant J. 2015;83:290–9.

    Article  CAS  PubMed  Google Scholar 

  29. Klein RR, Klein PE, Mullet JE, Minx P, Rooney WL, Schertz KF. Fertility restorer locus Rf1 of sorghum (Sorghum bicolor L.) encodes a pentatricopeptide repeat protein not present in the collinear region of rice chromosome 12. Theor Appl Genet. 2005;111:994–1012.

    Article  CAS  PubMed  Google Scholar 

  30. Hu J, Huang W, Huang Q, Qin X, Yu C, Wang L, Li S, Zhu R, Zhu Y. Mitochondria and cytoplasmic male sterility in plants. Mitochondrion. 2014;19:282–8.

    Article  CAS  PubMed  Google Scholar 

  31. Akagi H, Nakamura A, Yokozeki-Misono Y, Inagaki A, Takahashi H, Mori K, Fujimura T. Positional cloning of the rice rf-1 gene, a restorer of BT-type cytoplasmic male sterility that encodes a mitochondria-targeted PPR protein. Theor Appl Genet. 2004;108:1449–57.

    Article  CAS  PubMed  Google Scholar 

  32. Komori T, Ohta S, Murai N, Takakura Y, Kuraya Y, Suzuki S, Hiei Y, Imaseki H, Nitta N. Map-based cloning of a fertility restorer gene, Rf-1, in rice (Oryza sativa L.). Plant J. 2004;37:315–25.

    Article  CAS  PubMed  Google Scholar 

  33. Fujii S, Toriyama K. Suppressed expression of RETROGRADE_REGULATED MALE STERILITY restores pollen fertility in cytoplasmic male sterile rice plants. Proc Natl Acad Sci USA. 2009;106:9513–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Itabashi E, Iwata N, Fujii S, Kazama T, Toriyama K. The fertility restorer gene, Rf2, for Lead Rice-type cytoplasmic male sterility of rice encodes a mitochondrial glycine-rich protein. Plant J. 2011;65:359–67.

    Article  CAS  PubMed  Google Scholar 

  35. Fujii S, Bond CS, Small ID. Selection patterns on restorer-like genes reveal a conflict between nuclear and mitochondrial genomes throughout angiosperm evolution. Proc Natl Acad Sci USA. 2011;108:1723–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Dahan J, Mireau H. The Rf and Rf-like PPR in higher plants, a fast evolving subclass of PPR genes. RNA Biol. 2013;10:1286–93.

    Article  CAS  Google Scholar 

  37. Jo YD, Ha Y, Lee JH, Park M, Bergsma AC, Choi HI, Goritschnig S, Kloosterman B, van Dijk PJ. Fine mapping of Restorer‑of‑fertility in pepper (Capsicum annuum L.) identified a candidate gene encoding a pentatricopeptide repeat (PPR)‑containing protein. Theor Appl Genet. 2016;129: 2003–17.

    Article  CAS  PubMed  Google Scholar 

  38. Barchenger DW, Said JI, Zhang Y, Song M, Ortega FA, Ha Y, Kang BC, Bosland PW. Genome-wide identification of chile pepper pentatricopeptide repeat domains provides insight into fertility restoration. J Amer Soc Hort Sci. 2018;143:418–29.

    Article  Google Scholar 

  39. Novak F, Betlach J, Dubovsky J. Cytoplasmic malesterility in sweet pepper (Capsicum annuum L.) I. Phenotype and inheritance of male sterile character. Z Pflanzenzüchtung. 1971;65:129–40.

    Google Scholar 

  40. Wei B, Wang L, Chen L, Zhang R. Genetic analysis on the restoration of cytoplasmic male sterility with mixed model of major gene plus polygene in pepper. Acta Hort Sinica. 2013;40:2263–8.

    CAS  Google Scholar 

  41. Wei B, Wang L, Zhang R, Zhang J. Identification of two major quantitative trait loci restoring the fertility of cytoplasmic male sterility in Capsicum annuum. J Agric Biot. 2017;25:43–9.

    Google Scholar 

  42. Wang LH, Zhang BX, Lefebvre V, Huang SW, Daubèze AM, Palloix A. QTL analysis of fertility restoration in cytoplasmic male sterile pepper. Theor Appl Genet. 2004;109:1058–63.

    Article  CAS  PubMed  Google Scholar 

  43. Lee J, Yoon JB, Park HG. Linkage analysis between the partial restoration (pr) and the restorer-of-fertility (Rf) loci in pepper cytoplasmic male sterility. Theor Appl Genet. 2008;117:383–9.

    Article  CAS  PubMed  Google Scholar 

  44. Shifriss C. Male sterility in pepper (Capsicum annuum L.). Euphytica. 1997;93:83–8.

    Article  Google Scholar 

  45. Wang Z, Gerstein M, Snyder M. RNA-Seq: A revolutionary tool for transcriptomics. Nat Rev Genet. 2009;10:57–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Michelmore RW, Paran I, Kesseli RV. Identification of markers linked to disease-resistance genes by bulked segregate analysis – a rapid method to detect markers in specific genomic regions by using segregating populations. Proc Natl Acad Sci USA. 1991;88:9828–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Wang R, Sun L, Bao L, Zhang J, Jiang Y, Yao J, Song L, Feng J, Liu S, Liu Z. Bulk segregate RNA-seq reveals expression and positional candidate genes and allele specific expression for disease resistance against enteric septicemia of catfish. BMC Genomics. 2013;14:929.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Du H, Zhu J, Su H, Huang M, Wang H, Ding S, Zhang B, Luo A, Wei S, Tian X, Xu Y. Bulked segregate RNA-seq reveals differential expression and SNPs of candidate genes associated with water logging tolerance in maize. Front Plant Sci. 2017;8:1022.

    Article  PubMed  PubMed Central  Google Scholar 

  49. Castro AJ, Clement C. Sucrose and starch catabolism in the anther of Lilium during its development: a comparative study among the anther wall, locular fluid and microspore/pollen fractions. Planta. 2007;225:1573–82.

    Article  CAS  PubMed  Google Scholar 

  50. Rolland F, Moore B, Sheen J. Sugar sensing and signaling in plants. The Plant Cell. 2002;14:5185–205.

    Article  CAS  Google Scholar 

  51. Preuss D, Lemieux B, Yen G, Davis R. A conditional sterile mutation eliminates surface components from Arabidopsis pollen and disrupts cell signaling during fertilization. Genes Dev. 1993;7:974–85.

    Article  CAS  PubMed  Google Scholar 

  52. Rhee SY, Osborne E, Poindexter PD, Somerville CR. Microspore separation in the quartet mutant of Arabidopsis is impaired by a defect in a developmentally regulated polygalacturonase required for pollen mother cell wall degradation. Plant Physiol. 2003;133:1170–80.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Micheli F. Pectin methylesterases: cell wall enzymes with important roles in plant physiology. Trends Plant Sci. 2001;6:414–9.

    Article  CAS  PubMed  Google Scholar 

  54. Palusa SG, Golovkin M, Shin SB, Richardson DN, Reddy ASN. Organ-specific, developmental, hormonal and stress regulation of expression of putative pectate lyase genes in Arabidopsis. New Phytol. 2007;174:537–50.

    Article  CAS  PubMed  Google Scholar 

  55. Francis KE, Lam SY, Copenhaver GP. Separation of Arabidopsis pollen tetrads is regulated by QUARTET1, a pectin methylesterase gene. Plant Physiol. 2006;142:1004–13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Hamid R, Tomar RS, Marashi H, Shafaroudi SM, Golakiya BA, Mohsenpour M. Transcriptome profiling and cataloging differential gene expression in floral buds of fertile and sterile lines of cotton (Gossypium hirsutum L.). Gene. 2018;660:80–91.

    Article  CAS  PubMed  Google Scholar 

  57. Persson S, Paredez A, Carroll A, Palsdottir H, Doblin M, Poindexter P, Khitrov N, Auer M, Somerville CR. Genetic evidence for three unique components in primary cell-wall cellulose synthase complexes in Arabidopsis. Proc Natl Acad Sci USA. 2007;104:15566–71.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Wang W, Wang L, Chen C, Xiong G, Tan XY, Yang KZ, Wang ZC, Zhou Y, Ye D, Chen LQ. Arabidopsis CSLD1 and CSLD4 are required for cellulose deposition and normal growth of pollen tubes. J Exp Bot. 2011;62:5161–77.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Xue H, Chen X, Mei Y. Function and regulation of phospholipid signaling in plants. Biochem J. 2009;421:145–56.

    Article  CAS  PubMed  Google Scholar 

  60. Hamamura Y, Nagahara S, Higashiyama T. Double fertilization on the move. Curr Opin Plant Biol. 2012;15:70–7.

    Article  PubMed  Google Scholar 

  61. Chapman LA, Goring DR. Misregulation of phosphoinositides in Arabidopsis thaliana decreases pollen hydration and maternal fertility. Sex Plant Reprod. 2011;24:319–26.

    Article  CAS  PubMed  Google Scholar 

  62. Kost B, Lemichez E, Spielhofer P, Hong Y, Tolias K, Carpenter C, Chua NH. Rac homologues and compartmentalized phosphatidylinositol 4,5-bisphosphate act in a common pathway to regulate polar pollen tube growth. J Cell Biol. 1999;145:317–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Ischebeck T, Stenzel I, Heilmann I. Type B phosphatidylinositol-4-phosphate 5-kinases mediate Arabidopsis and Nicotiana tabacum pollen tube growth by regulating apical pectin secretion. Plt Cell. 2008;20:3312–30.

    Article  CAS  Google Scholar 

  64. Sousa E, Kost B, Malho R. Arabidopsis phosphatidylinositol-4-monophosphate 5-kinase 4 regulates pollen tube growth and polarity by modulating membrane recycling. Plant Cell. 2008;20:3050–64.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Ugalde JM, Rodriguez-Furlán C, Rycke R, Norambuena L, Friml J, León G, Tejos R. Phosphatidylinositol 4-phosphate 5-kinases 1 and 2 are involved in the regulation of vacuole morphology during Arabidopsis thaliana pollen development. Plant Sci. 2016;250:10–9.

    Article  CAS  PubMed  Google Scholar 

  66. Franklin-Tong VE. Signaling and the modulation of pollen tube growth. Plant Cell. 1999;11:727–38.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Paxson-Sowders DM, Owen HA, Makaroff CA. A comparative ultrastructural analysis of exine pattern development in wild-type Arabidopsis and a mutant defective in pattern formation. Protoplasma. 1996;198:53–65.

    Article  Google Scholar 

  68. Ahlers F, Thom I, Lambert J, Kuckuk R, Wiermann R. 1H NMR analysis of sporopollenin from Typha angustifolia. Phytochem. 1999;50:1095–8.

    Article  CAS  Google Scholar 

  69. Jiang J, Zhang Z, Cao J. Pollen wall development: the associated enzymes and metabolic pathways. Plant Biol. 2013;15:249–63.

    Article  CAS  PubMed  Google Scholar 

  70. Liu L, Fan XD. Tapetum: regulation and role in sporopollenin biosynthesis in Arabidopsis. Plant Mol Biol. 2013;83:165–75.

    Article  CAS  PubMed  Google Scholar 

  71. Ji J, Yang L, Fang Z, Zhuang M, Zhang Y, Lv H, Liu Y, Li Z. Complementary transcriptome and proteome profiling in cabbage buds of a recessive male sterile mutant provides new insights into male reproductive development. J Proteomics. 2018;179:80–91.

    Article  CAS  PubMed  Google Scholar 

  72. hoi H, Jin JY, Choi S, Hwang JU, Kim YY, Suh MC, Lee Y. An ABCG/WBC-type ABC transporter is essential for transport of sporopollenin precursors for exine formation in developing pollen. Plant J. 2011;65:181–93.

  73. Quilichini TD, Friedmann MC, Samuels AL, Douglas CJ. ATP-binding cassette transporter G26 is required for male fertility and pollen exine formation in Arabidopsis. Plant Physiol. 2010;154:678–90.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Wang K, Gao F, Ji Y, Liu Y, Dan Z, Yang P, Zhu Y, Li S. ORFH79 impairs mitochondrial function via interaction with a subunit of electron transport chain complex III in Honglian cytoplasmic male sterile rice. New Phytol. 2013;198:408–18.

    Article  CAS  PubMed  Google Scholar 

  75. Nie Z, Zhao T, Yang S, Gai J. Development of a cytoplasmic male-sterile line NJCMS4A for hybrid soybean production. Plant Breeding. 2017;136:516–25.

    Article  CAS  Google Scholar 

  76. Yang Y, Bao S, Zhou X, Liu J, Zhuang Y. The key genes and pathways related to male sterility of eggplant revealed by comparative transcriptome analysis. BMC Plant Biol. 2018;18:209–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Meech R, Mubarokah N, Shivasami A, Rogers A, Nair PC, Hu DG, McKinnon RA, Mackenzie PI. A novel function for UDP Glycosyltransferase 8: Galactosidation of bile acids. Mol Pharmacol. 2015;87:442–50.

    Article  PubMed  CAS  Google Scholar 

  78. Li X, Zhu B, Gao X, Liang P. Over-expression of UDP–glycosyltransferase gene UGT2B17 is involved in chlorantraniliprole resistance in Plutella xylostella (L.). Pest Manag Sci. 2017;73:1402–9.

    Article  PubMed  CAS  Google Scholar 

  79. Qin C, Yub C, Shena Y, Fang X, Chen L, Mind J, Cheng J, et al. Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization. Proc Natl Acad Sci USA. 2014;111:5135–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 2013;14:R36.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  81. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L. Transcript assembly and quantification by RNA Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010;28:511–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Rogers MF1, Thomas J, Reddy AS, Ben-Hur A. SpliceGrapher: detecting patterns of alternative splicing from RNA-Seq data in the context of gene models and EST data. Genome Biology. 2012;13:R4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Leng N, Dawson JA, Thomson JA, Ruotti V, Rissman AI, Smits BM, Haag JD, Gould MN, Stewart RM, Kendziorski C. EBSeq: An empirical Bayes hierarchical model for inference in RNA-seq experiments. Bioinformatics. 2013;29:1035–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Young MD, Wakefield MJ, Smyth GK, Oshlack A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 2010;11:R14.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  85. Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, Kawashima S, Okuda S, Tokimatsu T, Yamanishi Y. KEGG for linking genomes to life and the environment. Nucleic Acids Res. 2008;36:480–4.

    Article  CAS  Google Scholar 

  86. Mao X, Cai T, Olyarchuk JG, Wei L. Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary. Bioinformatics. 2005;21:3787–93.

    Article  CAS  PubMed  Google Scholar 

  87. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods. 2001;25:402–8.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Special thanks to Franchesca Ortega from the chile pepper breeding and genetics program at New Mexico State University for editing the manuscript.

Funding

This research was supported by the National Natural Sciences Foundation of China (31560555, 31760572). The part of high throughput sequencing and data analysis were srpported by the National Natural Sciences Foundation of China (31560555), and the qRT-PCR were commonly supported by the National Natural Sciences Foundation of China (31560555, 31760572).

Author information

Authors and Affiliations

Authors

Contributions

BW designed the experiments, developed the sterile pool and fertile pool, contributed to the bioinformation analysis and drafted the initial manuscript. LW provided the parental materials. PWB revised and edited the manuscript. GZ and RZ made the qRT-PCR analysis. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Bingqiang Wei.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Additional file 1: Table S1.

The KEGG Pathway related to the pepper restoration of fertility or pollen development.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wei, B., Wang, L., Bosland, P.W. et al. Comparative transcriptional analysis of Capsicum flower buds between a sterile flower pool and a restorer flower pool provides insight into the regulation of fertility restoration. BMC Genomics 20, 837 (2019). https://0-doi-org.brum.beds.ac.uk/10.1186/s12864-019-6210-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://0-doi-org.brum.beds.ac.uk/10.1186/s12864-019-6210-3

Keywords