Brassica vegetables exhibit pronounced heterosis; nevertheless, investigations on fertility-related genes are scarce. The present study scrutinized a recessive genic male-sterile line, 7-3A, capable of generating a completely sterile population, holding significant promise for flowering Chinese cabbage breeding. By whole-genome resequencing of sterile and fertile plants, the male-sterile gene was confined to approximately 185 kb on chromosome A07, situated between markers C719 and NP10 in Brassica rapa var. Chiifu-401. Notably, substantial structural variation was identified within this region across diverse Brassica rapa reference genomes. Despite discernible expression level disparities of a homologous gene, Bnams4b, between male sterile and fertile plants, no sequence divergence was detected. Further elucidation is required to pinpoint a novel sterile gene within the candidate interval. This investigation contributes to the advancement of both the molecular-assisted breeding scheme for flowering Chinese cabbage and the comprehension of male sterility mechanisms.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s13205-024-04005-7.

BACKGROUND: Long non-coding RNAs (lncRNAs) play a crucial role in regulating gene expression vital for the growth and development of plants. Despite this, the role of lncRNAs in Chinese cabbage (Brassica rapa L. ssp. pekinensis) pollen development and male fertility remains poorly understood.
RESULTS: In this study, we characterized a recessive genic male sterile mutant (366-2 S), where the delayed degradation of tapetum and the failure of tetrad separation primarily led to the inability to form single microspores, resulting in male sterility. To analyze the role of lncRNAs in pollen development, we conducted a comparative lncRNA sequencing using anthers from the male sterile mutant line (366-2 S) and the wild-type male fertile line (366-2 F). We identified 385 differentially expressed lncRNAs between the 366-2 F and 366-2 S lines, with 172 of them potentially associated with target genes. To further understand the alterations in mRNA expression and explore potential lncRNA-target genes (mRNAs), we performed comparative mRNA transcriptome analysis in the anthers of 366-2 S and 366-2 F at two stages. We identified 1,176 differentially expressed mRNAs. Remarkably, GO analysis revealed significant enrichment in five GO terms, most notably involving mRNAs annotated as pectinesterase and polygalacturonase, which play roles in cell wall degradation. The considerable downregulation of these genes might contribute to the delayed degradation of tapetum in 366-2 S. Furthermore, we identified 15 lncRNA-mRNA modules through Venn diagram analysis. Among them, MSTRG.9997-BraA04g004630.3 C (β-1,3-glucanase) is associated with callose degradation and tetrad separation. Additionally, MSTRG.5212-BraA02g040020.3 C (pectinesterase) and MSTRG.13,532-BraA05g030320.3 C (pectinesterase) are associated with cell wall degradation of the tapetum, indicating that these three candidate lncRNA-mRNA modules potentially regulate pollen development.
CONCLUSIONS: This study lays the foundation for understanding the roles of lncRNAs in pollen development and for elucidating their molecular mechanisms in regulating male sterility in Chinese cabbage.

Rice (Oryza sativa L.) is one of the most important food crops worldwide. The utilisation of heterosis (hybrid vigour) has played a significant role in increasing rice yield and ensuring food supply. Over the past 50 years, the first-generation three-line system based on cytoplasmic male sterility, and the second-generation two-line system based on environment-sensitive genic male sterility (EGMS), have been widely applied in hybrid rice production. However, the three-line system is restricted by the matching relationship among the three parental lines and allows only ~ 2-5% of germplasms to be explored for elite combinations. The environmental sensitivity of EGMS lines has posed serious risks to the production of hybrid seeds. These factors have hindered the development and applications of hybrid rice. Third-generation hybrid rice technology (TGHRT) is based on environment-insensitive genic male sterility, which can effectively overcome the intrinsic problems of the three-line and two-line systems. Since the establishment of TGHRT, numerous findings and innovations have been reported. This paper gives a brief review of traditional hybrid rice technologies and discusses the establishment of TGHRT, technical innovations in TGHRT, and future research that is necessary to promote the wide application of TGHRT in rice production.

[This corrects the article DOI: 10.3389/fpls.2018.01343.].

A novel male-sterility trait was identified in a radish (Raphanus sativus L.) population. Although the size of male-sterile anthers was comparable to that of normal flowers, no pollen grain was observed during anther dehiscence. However, dissection of male-sterile anthers revealed an abundance of normal pollen grains. Analysis of segregating populations showed that a single recessive locus, designated RsMs1, conferred male sterility. Based on two radish draft genome sequences, molecular markers were developed to delimit the genomic region harboring the RsMs1. The region was narrowed down to approximately 24 kb after analyzing recombinants selected from 7511 individuals of a segregating population. Sequencing of the delimited region yielded six putative genes including four genes expressed in the floral tissue, and one gene with significant differential expression between male-fertile and male-sterile individuals of a segregating population. This differentially expressed gene was orthologous to the Arabidopsis MYB26 gene, which played a critical role in anther dehiscence. Excluding a synonymous single nucleotide polymorphism in exon3, no polymorphism involving coding and putative promoter regions was detected between alleles. A 955-bp insertion was identified 7.5 kb upstream of the recessive allele. Highly conserved motifs among four Brassicaceae species were identified around this insertion site, suggesting the presence of putative enhancer sequences. A functional marker was developed for genotyping of the RsMs1 based on the 955-bp insertion. A total of 120 PI accessions were analyzed using this marker, and 11 accessions were shown to carry the recessive rsms1 allele.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s11032-021-01254-9.

BACKGROUND: Brassica napus is the third leading source of edible oil in the world. Genic male sterility (GMS) lines provide crucial material for harnessing heterosis for rapeseed. GMS lines have been used successfully for rapeseed hybrid production in China. MicroRNAs (miRNAs) play crucial regulatory roles in various plant growth, development, and stress response processes. However, reports on miRNAs that regulate the pollen development of GMS lines in B. napus are few.
RESULTS: In this study, 12 small RNA and transcriptome libraries were constructed and sequenced for the flower buds from the fertile and sterile lines of two recessive GMS (RGMS) lines, namely, \"6251AB\" and \"6284AB\". At the same time, 12 small RNA and transcriptome libraries were also constructed and sequenced for the flower buds from the fertile and sterile lines of two dominant GMS (DGMS) lines, namely, \"4001AB\" and \"4006AB\". Based on the results, 46 known miRNAs, 27 novel miRNAs on the other arm of known pre-miRNAs, and 44 new conserved miRNAs were identified. Thirty-five pairs of novel miRNA-3p/miRNA-5p were found. Among all the identified miRNAs, fifteen differentially expressed miRNAs with over 1.5-fold change between flower buds of sterile and fertile lines were identified, including six differentially expressed miRNAs between \"4001A\" and \"4001B\", two differentially expressed miRNAs between \"4006A\" and \"4006B\", four differentially expressed miRNAs between \"6251A\" and \"6251B\", and ten differentially expressed miRNAs between \"6284A\" and \"6284B\". The correlation analysis of small RNA and transcriptome sequencing was conducted. And 257 candidate target genes were predicted for the 15 differentially expressed miRNAs. The results of 5\' modified RACE indicated that BnaA09g48720D, BnaA09g11120D, and BnaCnng51960D were cleaved by bna-miR398a-3p, bna-miR158-3p and bna-miR159a, respectively. Among the differentially expressed miRNAs, miR159 was chosen to analyze its function. Overexpression of bna-miR159 in Arabidopsis resulted in decreased seed setting rate, and shortened siliques, illustrating that miR159 may regulate the fertility and silique development in rapeseed.
CONCLUSIONS: Our findings provide an overview of miRNAs that are potentially involved in GMS and pollen development. New information on miRNAs and their related target genes are provided to exploit the GMS mechanism and reveal the miRNA networks in B. napus.

Seed production is critical for watermelon production, which mostly involves first-generation hybrid varieties. However, watermelon hybrid seed production currently requires complex procedures, including artificial isolation and pollination. Therefore, the development and use of a male-sterile system to generate watermelon hybrids can simplify the process. The scarcity of male-sterile watermelon germplasm resources necessitates the use of molecular breeding methods. Unfortunately, the genes responsible for male sterility in watermelon have not been cloned. Thus, the genetic basis of the male sterility remains unknown. In this study, two DNA pools derived from male-sterile and normal plants in the F2 population were used for whole-genome resequencing. The Illumina high-throughput sequencing resulted in 62.99 Gbp clean reads, with a Q30 of 80% after filtering. On the basis of the SNP index association algorithm, eight candidate regions (0.32 Mb) related to specific traits were detected on chromosome 6. Expression pattern analyses and watermelon transformation studies generated preliminary evidence that Cla006625 encodes a pollen-specific leucine-rich repeat protein (ClaPEX1) influencing the male sterility of watermelon. The identification and use of genic male sterility genes will promote watermelon male sterility research and lay the foundation for the efficient application of seed production technology.

The utilization of heterosis is an important way to improve wheat yield, and the production of wheat hybrid seeds mainly relies on male-sterile lines. Male sterility in line 15 Fan 03 derived from a cross of 72,180 and Xiaoyan 6 is controlled by a single recessive gene. The gene was mapped to the distal region of chromosome 4BS in a genetic interval of 1.4 cM and physical distance of 6.57 Mb between SSR markers Ms4BS42 and Ms4BS199 using an F2 population with 1205 individuals. Sterile individuals had a deletion of 4.57 Mb in the region presumed to carry the Ms1 locus. The allele for sterility was therefore named ms1s. Three CAPS markers were developed and verified from the region upstream of the deleted fragment and can be used for ms1s marker-assisted selection in wheat hybrid breeding. This work will enrich the utilization of male sterility genetic resources.

The function and regulation of lipid metabolic genes are essential for plant male reproduction. However, expression regulation of lipid metabolic genic male sterility (GMS) genes by noncoding RNAs is largely unclear. Here, we systematically predicted the microRNA regulators of 34 maize white brown complex members in ATP-binding cassette transporter G subfamily (WBC/ABCG) genes using transcriptome analysis. Results indicate that the ZmABCG26 transcript was predicted to be targeted by zma-miR164h-5p, and their expression levels were negatively correlated in maize B73 and Oh43 genetic backgrounds based on both transcriptome data and qRT-PCR experiments. CRISPR/Cas9-induced gene mutagenesis was performed on ZmABCG26 and another lipid metabolic gene, ZmFAR1. DNA sequencing, phenotypic, and cytological observations demonstrated that both ZmABCG26 and ZmFAR1 are GMS genes in maize. Notably, ZmABCG26 proteins are localized in the endoplasmic reticulum (ER), chloroplast/plastid, and plasma membrane. Furthermore, ZmFAR1 shows catalytic activities to three CoA substrates in vitro with the activity order of C12:0-CoA > C16:0-CoA > C18:0-CoA, and its four key amino acid sites were critical to its catalytic activities. Lipidomics analysis revealed decreased cutin amounts and increased wax contents in anthers of both zmabcg26 and zmfar1 GMS mutants. A more detailed analysis exhibited differential changes in 54 monomer contents between wild type and mutants, as well as between zmabcg26 and zmfar1. These findings will promote a deeper understanding of miRNA-regulated lipid metabolic genes and the functional diversity of lipid metabolic genes, contributing to lipid biosynthesis in maize anthers. Additionally, cosegregating molecular markers for ZmABCG26 and ZmFAR1 were developed to facilitate the breeding of male sterile lines.

Fatty acyl reductases (FARs) catalyse the reduction of fatty acyl-coenzyme A (CoA) or -acyl carrier protein (ACP) substrates to primary fatty alcohols, which play essential roles in lipid metabolism in plants. However, the mechanism by which FARs are involved in male reproduction is poorly defined. Here, we found that two maize allelic mutants, ms25-6065 and ms25-6057, displayed defective anther cuticles, abnormal Ubisch body formation, impaired pollen exine formation and complete male sterility. Based on map-based cloning and CRISPR/Cas9 mutagenesis, Zm00001d048337 was identified as ZmMs25, encoding a plastid-localized FAR with catalytic activities to multiple acyl-CoA substrates in vitro. Four conserved residues (G101, G104, Y327 and K331) of ZmMs25 were critical for its activity. ZmMs25 was predominantly expressed in anther, and was directly regulated by transcription factor ZmMYB84. Lipidomics analysis revealed that ms25 mutation had significant effects on reducing cutin monomers and internal lipids, and altering the composition of cuticular wax in anthers. Moreover, loss of function of ZmMs25 significantly affected the expression of its four paralogous genes and five cloned lipid metabolic male-sterility genes in maize. These data suggest that ZmMs25 is required for anther development and male fertility, indicating its application potential in maize and other crops.