Sterility In Pepper Research Articles

Key candidate genes for male sterility in peppers unveiled via transcriptomic and proteomic analyses.

This study aimed to enhance the use of male sterility in pepper to select superior hybrid generations. Transcriptomic and proteomic analyses of fertile line 1933A and nucleic male sterility line 1933B of Capsicum annuum L. were performed to identify male sterility-related proteins and genes. The phylogenetic tree, physical and chemical characteristics, gene structure characteristics, collinearity and expression characteristics of candidate genes were analyzed. The study identified 2,357 differentially expressed genes, of which 1,145 and 229 were enriched in the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases, respectively. A total of 7,628 quantifiable proteins were identified and 29 important proteins and genes were identified. It is worth noting that the existence of CaPRX genes has been found in both proteomics and transcriptomics, and 3 CaPRX genes have been identified through association analysis. A total of 66 CaPRX genes have been identified at the genome level, which are divided into 13 subfamilies, all containing typical CaPRX gene conformal domains. It is unevenly distributed across 12 chromosomes (including the virtual chromosome Chr00). Salt stress and co-expression analysis show that male sterility genes are expressed to varying degrees, and multiple transcription factors are co-expressed with CaPRXs, suggesting that they are involved in the induction of pepper salt stress. The study findings provide a theoretical foundation for genetic breeding by identifying genes, metabolic pathways, and molecular mechanisms involved in male sterility in pepper.

Characterization of Plant Homeodomain Transcription Factor Genes Involved in Flower Development and Multiple Abiotic Stress Response in Pepper.

Plant homeodomain (PHD) transcription factor genes are involved in plant development and in a plant's response to stress. However, there are few reports about this gene family in peppers (Capsicum annuum L.). In this study, the pepper inbred line "Zunla-1" was used as the reference genome, and a total of 43 PHD genes were identified, and systematic analysis was performed to study the chromosomal location, evolutionary relationship, gene structure, domains, and upstream cis-regulatory elements of the CaPHD genes. The fewest CaPHD genes were located on chromosome 4, while the most were on chromosome 3. Genes with similar gene structures and domains were clustered together. Expression analysis showed that the expression of CaPHD genes was quite different in different tissues and in response to various stress treatments. The expression of CaPHD17 was different in the early stage of flower bud development in the near-isogenic cytoplasmic male-sterile inbred and the maintainer inbred lines. It is speculated that this gene is involved in the development of male sterility in pepper. CaPHD37 was significantly upregulated in leaves and roots after heat stress, and it is speculated that CaPHD37 plays an important role in tolerating heat stress in pepper; in addition, CaPHD9, CaPHD10, CaPHD11, CaPHD17, CaPHD19, CaPHD20, and CaPHD43 were not sensitive to abiotic stress or hormonal factors. This study will provide the basis for further research into the function of CaPHD genes in plant development and responses to abiotic stresses and hormones.

Development of KASP molecular markers related to the fertility restoration of cytoplasmic male sterility in pepper

Cytoplasmic male sterility (CMS) and Restorer-of-fertility (Rf) breeding systems can save the cost of the production of F1 hybrid seed in crops. However, the breeding of the restorer lines is not easy and the current molecular markers related to Rf gene is not very versatile because of diverse genes regulating the restoration. In this study, 11 SNPs in the previous Rf gene mapping region near the 3’ end of chromosome 6 of pepper were used to develop the competitive allele-specific PCR (KASP) molecular markers. The results showed that KS23 was proposed to relate to the Rf genes with the restoring genotype A/A and the maintaining genotype G/G. There were 91.6% in consistency for KS23 between the genotyping results in F2 individuals. In addition, the accuracy of KS23 achieved 100% among six restorer lines and 67.8% among 44 inbred lines in universalities, respectively. These results provided an efficient and labor saving KASP marker related to Rf, which will be beneficial for the breeding of restorer lines and accelerate the utility of CMS/Rf system in the production of F1 hybrid seed in pepper.

Orf165 is associated with cytoplasmic male sterility in pepper.

Cytoplasmic male sterility (CMS) is a maternally inherited trait that derives from the inability to produce functional pollen in higher plants. CMS results from recombination of the mitochondrial genome. However, understanding of the molecular mechanism of CMS in pepper is limited. In this study, comparative transcriptomic analyses were performed using a near-isogenic CMS line 14A (CMS-14A) and a maintainer line 14B (ML-14B) as experimental materials. A total of 17,349 differentially expressed genes were detected between CMS-14A and ML-14B at the PMC meiosis stage. Among them, six unigenes associated with CMS and 108 unigenes involved in energy metabolism were identified. The gene orf165 was found in CMS-14A. When orf165 was introduced into ML-14B, almost 30% of transgenic plants were CMS. In addition, orf165 expression in transgenic CMS plants resulted in abnormal function of some genes involved in energy metabolism. When orf165 in transgenic CMS plant was silenced, the resulted orf165-silenced plant was male fertile and the expression patterns of some genes associated with energy metabolism were similar to ML-14B. Thus, we confirmed that orf165 influenced CMS in pepper.

Systematic identification and characterization of long non-coding RNAs involved in cytoplasmic male sterility in pepper (Capsicum annuum L.)

Long non-coding RNAs (lncRNAs) participate in many biological processes and play extremely important regulatory roles in animals and plants. However, little is known about lncRNAs and their functions in cytoplasmic male sterility (CMS) during the developmental process of floral buds in pepper. In the present study, we systematically identified lncRNAs in the pepper CMS line (9704A) and maintainer line (9704B) using high-throughput sequencing and bioinformatic analyses. A total of 10,655 putative lncRNAs were identified during the developmental process of floral buds, and 262, 298 and 905 differentially expressed lncRNAs (DELs) were obtained in three stages, respectively. Moreover, 1137 unique DELs were identified between 9704A and 9704B, and their cis and trans target protein-coding genes were predicted. We used Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses to decipher the potential functions of DELs. The cell and cellular process GO terms were significantly enriched, and KEGG pathway analysis showed enrichments in the pathways for starch and sucrose metabolism genes, pentose and glucuronate interconversions, all of which were highly associated with pollen development. Besides, a regulatory network about the lncRNAs and the protein-coding genes involved in the formation of CMS was constructed. Our findings laid a foundation for further studies on the function of lncRNAs in pollen development and provided valuable insights into the regulatory mechanism underlying CMS.

Comparative Transcriptome Analysis between Cytoplasmic Male-sterile Line and Its Maintainer During the Floral Bud Development of Pepper

The male-sterile line has been largely used in the hybrid seed production of pepper, which can effectively improve the efficiency of hybrid seed production. However, the formation mechanism of male sterility in pepper remains unclear. In the present study, we compared the gene expression patterns between pepper cytoplasmic male sterile line 9704A and its maintainer 9704B during floral bud development using RNA sequencing technology. A total of 547 976 and 2 416 Differentially Expressed Genes (DEGs) were identified in the stage S1, S2 and S3, respectively, and more than 70% of the DEGs were down-regulated in the sterile line. Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to further understand the functions of these identified DEGs. The results showed that the DEGs were mainly enriched in pathways of starch and sucrose metabolism, pentose and glucuronate interconversions. A number of genes, such as MS1, PME5, ATPB, and lots of transcription factors were found down-regulated in the sterile line, and we also identified a series of genes with large differences in expression patterns between sterile line and maintainer line. Collectively, our findings laid a foundation for further molecular breeding in pepper and provided new insights into its mechanism underlying the male sterility.

A Joint Segregation Analysis of the Inheritance of Fertility Restoration for Cytoplasmic Male Sterility in Pepper

A cytoplasmic male sterility (CMS) system is one of the most efficient ways to produce F1 hybrid seeds in pepper (Capsicum annuum). Restorer-of-fertility (Rf) genes are a critical component within the CMS/Rf system. The inheritance of Rf genes in pepper by joint segregation analysis was examined. The inheritance of Rf genes in the two progenies was controlled by two major additive-dominant epistatic genes and additive-dominant epistasis polygene. The two major genes had high additive effects and dominant effects. In addition, there existed significant epistatic effects between the two major genes. The major genes had high heritability in F2, BC1, and BC2 generations. Also, the fertility restorer characteristic can be selected during early generations of the breeding cycle.

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.

Proteomic analysis reveals strong mitochondrial involvement in cytoplasmic male sterility of pepper (Capsicum annuum L.)

Although cytoplasmic male sterility (CMS) is widely used for developing pepper hybrids, its molecular mechanism remains unclear. In this study, we used a high-throughput proteomics method called label-free to compare protein abundance across a pepper CMS line (A-line) and its isogenic maintainer line (B-line). Data are available via ProteomeXchange with identifier PXD006104. Approximately 324 differentially abundant protein species were identified and quantified; among which, 47 were up-accumulated and 140 were down-accumulated in the A-line; additionally, 75 and 62 protein species were specifically accumulated in the A-line and B-line, respectively. Protein species involved in pollen exine formation, pyruvate metabolic processes, the tricarboxylic acid cycle, the mitochondrial electron transport chain, and oxidative stress response were observed to be differentially accumulated between A-line and B-line, suggesting their potential roles in the regulation of pepper pollen abortion. Based on our data, we proposed a potential regulatory network for pepper CMS that unifies these processes. Biological significanceArtificial emasculation is a major obstacle in pepper hybrid breeding for its high labor cost and poor seed purity. While the use of cytoplasmic male sterility (CMS) in hybrid system is seriously frustrated because a long time is needed to cultivate male sterility line and its isogenic restore line. Transgenic technology is an effective and rapid method to obtain male sterility lines and its widely application has very important significance in speeding up breeding process in pepper. Although numerous studies have been conducted to select the genes related to male sterility, the molecular mechanism of cytoplasmic male sterility in pepper remains unknown. In this study, we used the high-throughput proteomic method called “label-free”, coupled with liquid chromatography-quadrupole mass spectrometry (LC–MS/MS), to perform a novel comparison of expression profiles in a CMS pepper line and its maintainer line. Based on our results, we proposed a potential regulated protein network involved in pollen development as a novel mechanism of pepper CMS.

Involvement of a universal amino acid synthesis impediment in cytoplasmic male sterility in pepper.

To explore the mechanisms of pepper (Capsicum annuum L.) cytoplasmic male sterility (CMS), we studied the different maturation processes of sterile and fertile pepper anthers. A paraffin section analysis of the sterile anthers indicated an abnormality of the tapetal layer and an over-vacuolization of the cells. The quantitative proteomics results showed that the expression of histidinol dehydrogenase (HDH), dihydroxy-acid dehydratase (DAD), aspartate aminotransferase (ATAAT), cysteine synthase (CS), delta-1-pyrroline-5-carboxylate synthase (P5CS), and glutamate synthetase (GS) in the amino acid synthesis pathway decreased by more than 1.5-fold. Furthermore, the mRNA and protein expression levels of DAD, ATAAT, CS and P5CS showed a 2- to 16-fold increase in the maintainer line anthers. We also found that most of the amino acid content levels decreased to varying degrees during the anther tapetum period of the sterile line, whereas these levels increased in the maintainer line. The results of our study indicate that during pepper anther development, changes in amino acid synthesis are significant and accompany abnormal tapetum maturity, which is most likely an important cause of male sterility in pepper.

Identification of proteins associated with cytoplasmic male sterility in pepper (Capsicum annuum L.)

Pepper (Capsicum annuum L.) is a globally important and often cross-pollinated vegetable that exhibits significant heterosis. Though cytoplasmic male sterility (CMS) has an important value for pepper hybrid production, its molecular mechanism remains unclear. To explore the molecular mechanism of CMS in pepper at the proteome level, we examined the anther proteomes of a cytoplasmic male sterile line FS1030A and its maintainer line FS1030B by transmission electronic microscopy (TEM) during abortion at the microspore mother cell stage. Two dimension difference gel electrophoresis (2D-DIGE) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) analyses were used to determine differentially expressed proteins. About 1070 anther protein spots were detected on each gel, and 13 protein spots were expressed differently between FS1030A and FS1030B (p<0.05). Twelve of the thirteen protein spots (92.3%) were successfully identified by NCBInr as nine proteins, namely down-regulated triosephosphate isomerase, actin, glutathione S-transferase (GST), Cu/Zn superoxide dismutase (Cu/Zn-SOD), and up-regulated branched-chain α-keto acid dehydrogenase E3 subunit, ketol-acid reductoisomerase (KARI), aspartic protease, and nascent polypeptides. The low-expression of triosephosphate isomerase involved in glycolysis impacts oxidative phosphorylation significantly and ultimately results in a reduction in ATP production, and over-expression of aspartic protease blocking normal programmed cell death (PCD) played important roles in the occurrence of CMS-FS1030A. This is the first study on pepper CMS using a proteomic approach. These findings provide a good basis for further studies of CMS in pepper.

A CMS-Related Gene, Ψatp6-2, Causes Increased ATP Hydrolysis Activity of the Mitochondrial F1Fo-ATP Synthase and Induces Male Sterility in Pepper (Capsicum annuum L.)

Pollen formation is a complex process that is strictly controlled by genetic factors. Although many novel mitochondrial genes have been implicated in the dysfunction of mitochondrial enzymes and the cytoplasmic male sterility (CMS), there is little empirical evidence to show that CMS-related genes actually result in the dysfunction of enzyme and little is known about the regulatory mechanisms of the aberrant mitochondrial enzymes in male sterility in CMS lines. Here, we report the characterization of a novel mitochondrial gene, Ψatp6-2, which is hypothesized to play a role in male sterility in pepper. Using virus-induced gene silencing (VIGS), we observed that silencing the atp6-2 gene in the maintainer line resulted in an increase in ATP hydrolysis activity of the mitochondrial F1Fo-ATP synthase along with pollen abortion, while silencing the truncated Ψatp6-2 gene in the CMS line resulted in an inhibition of ATP hydrolysis activity and restoration of fertility. Altered ATP hydrolysis also affected the tolerance of the gene-silenced plants to abiotic stresses. Localization experiments showed that premature ATP hydrolysis occurred at the tetrad stage of pollen development in the CMS line, but no ATPase activity was observed in the microspores at the later stage. These results suggest that the Ψatp6-2 gene causes the alteration in ATP hydrolysis activity of the mitochondrial F1Fo-ATP synthase during pollen development, which eventually leads to male sterility in pepper.

Induction and exploitation of nuclear and cytoplasmic male sterility in pepper (Capsicum spp.): a review

SummaryPepper belongs to the genus Capsicum, of which five species are cultivated (C. annuum L., C. chinense Jacq., C. frutescens L., C. baccatum L. Ruiz. & Pavon, and C. pubescens). C. annuum L., which includes both hot pepper and bell pepper, is the most widely cultivated species. Commercial hybrid pepper seed is produced manually or by exploiting male sterility (MS). Both nuclear (NMS) and cytoplasmic (CMS) MS are available in pepper. Due to recessive gene control, the development of NMS lines through conventional breeding is tedious and time-consuming. Molecular markers linked to the NMS genes ms-1 and ms-8 have been developed. This will facilitate the development of new NMS lines, identify MS plants at the seedling stage, and test the purity of F1 hybrid seed. To reduce hybrid seed costs further, and to improve seed yields, there is a need to identify seedling markers to facilitate the removal of fertile plants in nursery blocks. Most reports have indicated that CMS is sensitive to low temperatures. Restorer-of-fertility genes are common in hot pepper and rare in bell pepper cultivars. Despite conflicting reports, it has been concluded that a single dominant gene (Rf) along with a modifier gene (Pr), controls the restoration of fertility in pepper. Molecular markers linked to the Rf and Pr genes and the mitochondrial CMS genes coxII and atp6 have been developed. These will facilitate the rapid screening of inbred pepper lines for Rf/rf alleles, accelerate the transfer of the rf gene in female hot pepper parents (maintainer breeding), and the Rf gene in male sweet pepper parents (restorer breeding). We have compiled up-to-date information on both NMS and CMS systems in pepper and discuss the prospects for their use in breeding for heterosis.

Development of a SCAR marker for early identification of S-cytoplasm based on mitochondrial SRAP analysis in pepper (Capsicum annuum L.)

Molecular markers, coxII SCAR, atp6-2 SCAR and accD-U, have been used for marker-assisted selection of cytoplasmic male sterility (CMS) in pepper. However, the presence of these markers at the sub-stoichiometric level in maintainer lines affects the reliable selection of male sterile (S-) cytoplasm. This study aimed to develop a new CMS-specific molecular marker, SCAR130, for reliable identification of S-cytoplasm in pepper, while the new and three previous molecular markers were used to determine the cytoplasm types of pepper lines. Based on mitochondrial genome sequence related amplified polymorphism (SRAP) analysis of the CMS lines and the maintainer lines, SCAR130 was developed from a 10-bp deletion at the SRAP primer binding site in the CMS line (130 bp) compared with that in the maintainer line (140 bp). S-cytoplasm could be unambiguously selected from the pepper lines by the different length of the marker bands. Application of the four molecular markers to various pepper lines revealed that SCAR130 is more reliable than the other three previous markers, orf507, ψatp6-2 and accD-U. Homology alignment with BLAST showed that the marker was located between trnE and trnS in the Nicotiana tabacum mitochondrial genome. Furthermore, expression of the marker-linked gene was significantly higher at the pollen abortive stage in the CMS line (HW203A) than in the maintainer line, which indicated that the marker was closely related to male sterility. Hence, factors other than orf507 and ψatp6-2 may exist for the regulation of male sterility in pepper.

Characterization and inheritance of a novel thermo-sensitive restoration of cytoplasmic male sterility in Capsicum annuum

Thermo-sensitive male sterility can be used for two-line hybrid production, if the fertility can be manipulated by temperature shifting. In this study, we investigated a novel thermo-sensitive cytoplasmic male-sterile (TCMS) line of pepper. The TCMS line was sterile at temperatures above 15°C but their fertility was restored when night temperatures were below 13°C. To elucidate the cellular mechanism acting in the TCMS line, histological analysis was performed using light microscopy and transmission electron microscopy (TEM). We demonstrated that stable male sterility in pepper might be induced due to the abnormal development of tapetal cells whereas defects of callose wall biosynthesis seemed to be the main reason for male sterility in TCMS plants. Meiotic cell division in sterile TCMS plants proceeded to the last stage, whereas normal sterile plants were arrested at the meiotic stage. Further analysis using TEM revealed that abnormal development and failure of timely programmed cell death of the tapetum led to secretory dysfunction of tapetal cells. Consequently, this secretory defect may have influenced callose wall deposition and pollen wall formation in sterile TCMS. To reveal genetic mechanisms influencing TCMS, inheritance and marker analysis was performed. Cytoplasmic male sterility (CMS)-associated marker tests showed that the TCMS line contains CMS cytoplasm. Inheritance patterns and Rf-linked marker analysis suggested that TCMS is controlled by an allele named RfTCMS located at the Rf locus. Restoring experiment at low temperature showed that the thermo-sensitive restorer, RfTCMS, may be another allele located on the Rf locus and is recessive to Rf and dominant to rf.

Reduced activity of ATP synthase in mitochondria causes cytoplasmic male sterility in chili pepper

Cytoplasmic male sterility (CMS) is a maternally inherited trait characterized by the inability to produce functional pollen. The CMS-associated protein Orf507 (reported as Orf456 in previous researches) was previously identified as a candidate gene for mediating male sterility in pepper. Here, we performed yeast two-hybrid analysis to screen for interacting proteins, and found that the ATP synthase 6kDa subunit containing a mitochondrial signal peptide (MtATP6) specifically interacted with Orf507. In addition, the two proteins were found to be interacted in vivo using bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) assays. Further functional characterization of Orf507 revealed that the encoded protein is toxic to bacterial cells. Analysis of tissue-specific expression of ATP synthase 6kDa showed that the transcription level was much lower in anthers of the CMS line than in their wild type counterparts. In CMS plants, ATP synthase activity and content were reduced by more than half compared to that of the normal plants. Taken together, it can be concluded that reduced ATP synthase activity and ATP content might have affected pollen development in CMS plants. Here, we hypothesize that Orf507 might cause MtATP6 to be nonfunctional by changing the latter's conformation or producing an inhibitor that prevents the normal functioning of MtATP6. Thus, further functional analysis of mitochondrial Orf507 will provide insights into the mechanisms underlying CMS in plants.

Relationship of metabolism of reactive oxygen species with cytoplasmic male sterility in pepper ( Capsicum annuum L.)

Pepper cytoplasmic male sterility (CMS) line 9704A is one of the CMS types used for hybrid pepper ( Capsicum annuum L.) production in China. Our previous studies suggested that CMS-9704A may suffer from oxidative stress as its cyanide-resistant respiration is lower than that of the maintainer line. To elucidate the metabolic mechanism of reactive oxygen species (ROS) in the CMS-pepper anthers, the metabolism changes in the production and scavenging of ROS and gene expression related to ROS-scavenging enzymes were investigated in the anther mitochondria of CMS-9704A and maintainer-9704B. At the abortion stage (pollen mother cell meiosis stage), anthers of CMS-9704A had higher contents of O 2 −, H 2O 2 and MDA than those of the maintainer. Simultaneously, there were higher activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in scavenging ROS in the anthers of the CMS line than in the maintainer. Abundance of POD, Mn-SOD, CAT1, CAT2 and CAT3 mRNA in anthers of CMS-9704A corresponded with the activities of these three genes, except for that of Cu/Zn-SOD and Irion-SOD. Therefore, it is possible that the sterility in CMS-9704A is related to the abortion of microspores, induced by chronic oxidative stress caused by an abnormal increase in ROS, and also by membrane lipid peroxidation in the mitochondria in the early stage of pollen development.

Development of a CMS-specific marker based on chloroplast-derived mitochondrial sequence in pepper

Molecular markers developed from the flanking sequences of two cytoplasmic male sterility (CMS)-associated genes, orf456 and ψatp6-2, have been used for marker-assisted selection of CMS in pepper. However, in practice, the presence of orf456 and ψatp6-2 at substoichiometric levels even in maintainer lines hampers reliable selection of plants containing the CMS gene. In this study, we developed a novel CMS-specific molecular marker, accD-U, for reliable determination of CMS lines in pepper, and used the newly and previously developed markers to determine the cytoplasm types of pepper breeding lines and germplasms. This marker was developed from a deletion in a chloroplast-derived sequence in the mitochondrial genome of a CMS pepper line. CMS pepper lines could be unambiguously determined by presence or absence of the accD-U marker band. Application of orf456, ψatp6-2 and accD-U to various pepper breeding lines and germplasms revealed that accD-U is the most reliable CMS selection marker. A wide distribution of orf456, but not ψatp6-2, in germplasms suggests that the pepper cytoplasm containing both orf456 and ψatp6-2 has been selected as CMS cytoplasm from cytoplasm containing only orf456. Furthermore, factors other than orf456 may be required for the regulation of male sterility in pepper.

Genetics of Fertility Restoration in Cytoplasmic Male Sterile Pepper

Abstract Pepper hybrid seeds production using male sterility could lower cost by reducing time and labour, and increase the genetic purity of the F 1 seeds. To investigate the genetics of fertility restoration of the Peterson cytoplasmic sterility in pepper, a doubled haploid population of 115 pepper lines obtained from anther culture of the F 1 hybrid between Yolo Wonder (sterility maintainer line) and Perennial (fertility restorer line) and the parental lines were test-crossed by 77013A (a strict cytoplasmic-genic male sterile line). The fertility of the test-crossed lines was assessed in greenhouse and open field with the following three criteria: pollen index (PI, visual estimation of pollen amount per flower), pollen number (PN, pollen counting under microscope), and seed number (SN, the number of seeds per fruit in open pollination). Correlations between the each couple of criteria within, as well as between the cultivation methods ranged from 0.55 to 0.84. Analysis of variance showed that the genotype (DH line) and environment were the significant sources of variation of the fertility. Narrow sense of heritance of fertility restoration ranged from 0.38 to 0.92, depending on the criteria and environment. The distribution of the progeny was continuous between the parental genotypes indicating the quantitative inheritance of fertility restoration. Inferred from segregation according to Snape et al.(1984), the number of segregating genes was estimated to be that three to four genetic factors were involved in pollen traits (PI and PN) and five to eight genetic factors in seed production (SN). The heredity analysis of the CMS will be helpful for understanding of the genetic mechanism of the fertility restoration and the exploitation of the CMS in hybrid seed production.

Digenic nature of male sterility in pepper (Capsicum annuum L.)

A cross was made between two nearly isogenic lines differing for male sterility genes, viz. ms1ms1Ms2Ms2 s Ms1Ms1Ms2ms2. F1 plants yielded F2 populations which segregated either in 3:1 or 9:7 ratios of fertile vs male sterile individuals. Test crosses between male sterile and male fertile sibs in the 9:7 segregating populations provided a few lines in which most of the progenies were male sterile. A 3:1 ratio model of male steriles vs fertiles is suggested and the value of the system is discussed.