Genome-wide identification and expression analysis of the 2OG-Fe(II) oxygenase gene family in upland cotton (Gossypium hirsutum L.)

The 2OG-Fe(II) oxygenase (RF) family of enzyme proteins can affect bulliform cells and cause leaf curling. However, there are few studies related to this family in cotton, and there has been no systematic analysis of RF genes. Here, we determined 25 RF genes in the complete genome sequence of upland cotton (Gossypium hirsutum L.) and 11 RF genes in the complete genome sequence of Arabidopsis thaliana. Cotton RF proteins can be divided into three categories. Whole genome/fragment and scattered replication events played an important role in the expansion of the RF gene family. qRT-PCR analysis results showed that RF genes respond to drought stress Pairwise comparison results showed that the expression of RF genes in Shi yuan 321 was higher than that in Kui 85–174. Overall, genome-wide identification approach was used to further analyze the related functions of the RF gene family, which may include the response to drought stress, in cotton.

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

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

Mapping and validation of markers linked to Rf gene in sweet pepper (Capsicum annuum var grossum)

Cytoplasmic male sterility (CMS), is the most valuable system in commercial hybrid seed production in hot pepper. Whereas in sweet pepper (Capsicum annuum var grossum), the unavailability of a stable restorer’s line is one of the major constraints in commercial hybrid breeding. Identifying the markers linked to CMS and nuclear restorer-of-fertility (Rf) genes will help in the production of CMS hybrids in sweet pepper. In the present study, we identified seven markers co-segregating with Rf genes using bulk segregating analysis (BSA) on the F2 Rf-segregating mapping population. Among these, the CRF-SCAR marker proved to be linked to the fertility restorer gene and it could be used for screening of genotypes to identify restorers and non-restorer lines in future Capsicum breeding programs and help in the establishment of CMS system for commercial hybrid seed production.

A predicted NEDD8 conjugating enzyme gene identified as a Capsicum candidate Rf gene using bulk segregant RNA sequencing

Cytoplasmic male sterility (CMS) is an important tool for producing F1 hybrids, which can exhibit heterosis. The companion system, restorer-of-fertility (Rf), is poorly understood at the molecular level and would be valuable in producing restorer lines for hybrid seed production. The identity of the Rf gene in Capsicum (pepper) is currently unclear. In this study, using bulked segregant RNA sequencing (BSR-seq), a strong candidate Rf gene, Capana06g002866, which is annotated as a NEDD8 conjugating enzyme E2, was identified. Capana06g002866 has an ORF of 555 bp in length encoding 184 amino acids; it can be cloned from F1 plants from the hybridization of the CMS line 8A and restorer line R1 but is not found in CMS line 8A. With qRT-PCR validation, Capana06g002866 was found to be upregulated in restorer accessions compared to sterile accessions. The relative expression in flower buds increased with the developmental stage in F1 plants, while the expression was very low in all flower bud stages of the CMS lines. These results provide new insights into the Rf gene in pepper and will be useful for other crops utilizing the CMS system.

About the creation of sterile lines of sweet pepper in breeding for heterosis

Relevance. Development of valuable sterile lines of sweet pepper using nuclear-cytoplasmic male sterility is associated with the search for stable sterility maintainers in collection material.Material and methods. The purpose of the research is to optimize the identification of the desired genotypes and accelerate the development of sterile lines using artificial climate chambers. The research material was a sterile line msTol55, which under the conditions of a film greenhouse showed partial fertility at the beginning or at the end of the growing season. The place of cultivation is artificial climate chamber, a film spring greenhouse.50-day-old seedlings were planted in pots on 10.01, the period of growing plants in the chamber - until 20.04, then the adult plants were transplanted into a spring film greenhouse. According to the traditional technology, the cassette seedlings of the same line were planted in the greenhouse on 15.04. The number of tested plants is 16-20 pcs. Growing mode in the chamber: 12 hours were illuminated with DRLV lamps, while the temperature was at the level of 26-28°C, from 20.00 to 8.00 the lamps were not illuminated, the temperature was 14-15°C. The actual sterility / fertility of the flowers was determined by the level of contamination of the fruits at the beginning of their formation.Results. The maintained temperature regime for growing in an artificial climate chamber made it possible to identify sterile (30%) and partially fertile plants (70%) in the first 2 weeks of flowering. When adult plants were transplanted into a spring greenhouse, the number of sterile plants increased to 56%. When growing the line msTol55 in a spring greenhouse using the usual technology, partial setting of seeded and little seeded fruits on 60% of the plants was observed from the third decade of May to the second decade of July, then all plants set only non-seeded fruits. Based on the results obtained, it is advisable to identify the breeding material for the Rf gene in the ACC, since the proposed growing regime serves as a provocative background and allows for more effective control over the manifestation of sterility.

Genome-Wide Correlation of 36 Agronomic Traits in the 287 Pepper (Capsicum) Accessions Obtained from the SLAF-seq-Based GWAS

There are many agronomic traits of pepper (Capsicum L.) with abundant phenotypes that can benefit pepper growth. Using specific-locus amplified fragment sequencing (SLAF-seq), a genome-wide association study (GWAS) of 36 agronomic traits was carried out for 287 representative pepper accessions. To ensure the accuracy and reliability of the GWAS results, we analyzed the genetic diversity, distribution of labels (SLAF tags and single nucleotide polymorphisms (SNPs)) and population differentiation and determined the optimal statistical model. In our study, 1487 SNPs were highly significantly associated with 26 agronomic traits, and 2126 candidate genes were detected in the 100-kb region up- and down-stream near these SNPs. Furthermore, 13 major association peaks were identified for 11 key agronomic traits. Then we examined the correlations among the 36 agronomic traits and analyzed SNP distribution and found 37 SNP polymerization regions (total size: 264.69 Mbp) that could be selected areas in pepper breeding. We found that the stronger the correlation between the two traits, the greater the possibility of them being in more than one polymerization region, suggesting that they may be linked or that one pleiotropic gene controls them. These results provide a theoretical foundation for future multi-trait pyramid breeding of pepper. Finally, we found that the GWAS signals were highly consistent with those from the nuclear restorer-of-fertility (Rf) gene for cytoplasmic male sterility (CMS), verifying their reliability. We further identified Capana06g002967 and Capana06g002969 as Rf candidate genes by functional annotation and expression analysis, which provided a reference for the study of cytoplasmic male sterility in Capsicum.

Early in vitro selection of winter oilseed rape (Brassica napus L.) plants with the fertility restorer gene for CMS Shaan 2A via non-destructive molecular analysis of microspore-derived embryos

Cotyledons of 201 microspore-derived oilseed rape (Brassica napus) embryos were analysed for the presence of the cytoplasmic male sterility Shaan 2A restorer gene using the primer pair 3F/4R. In total, 172 plants regenerated and were grown to the generative stage. Among phenotypically non-haploid flowering plants, there was a broad spectrum of different flower types from normally developed flowers to flowers with short anthers, stamens, or deformed flowers with atypically developed floral organs, often with traces of pollen. For this reason, only 115 typically fertile or sterile accessions were selected for further phenotype-genotype comparisons. In 14 plants (12.2%) the phenotype did not match the genotype: two plants were marked as fertile, but without the Rf gene. Vice versa, in 12 sterile plants the Rf gene was determined. Nevertheless, the two fertile plants did not produce any pods after self-pollination. In total, 47 doubled haploid (DH) lines with good seed yield, carrying the Rf gene were harvested.