Intraspecific variations of the cytoplasmic male sterility genes orf108 and orf117 in Brassica maurorum and Moricandia arvensis, and the specificity of the mRNA processing

The mitochondrial gene <i>orf108</i> co-transcribed with <i>atp1</i> and causes cytoplasmic male sterility in <i>Brassica</i> crops, is widely distributed across wild species and genera of <i>Brassicaceae</i>. However, intraspecific variations in the presence of <i>orf108</i> have not yet been studied, and the mechanisms for the wide distribution of the gene remain unclear. We analyzed the presence and sequence variations of <i>orf108</i> in two wild species, <i>Brassica maurorum</i> and <i>Moricandia arvensis</i>. After polymerase chain reaction amplification of the 5′ region of <i>atp1</i> and the coding sequence of <i>orf108</i>, we determined the DNA sequences. <i>B. maurorum</i> and <i>M. arvensis</i> showed variations for the presence of <i>orf108</i> or <i>orf117</i> (<i>orf108^V117</i>) both between and within accessions, and were not fixed to the mitochondrial type having the male sterile genes. Sequencing of the amplicons clarified that <i>B. maurorum</i> has <i>orf108^V117</i> instead of <i>orf108</i>. Sequencing also indicated mitochondrial heteroplasmy in the two species; particularly, in <i>B. maurorum</i>, one plant possessed both the <i>orf108</i> and <i>orf108^V117</i> sequences. The results suggested that substoichiometric shifting of the mitochondrial genomes leads to the acquisition or loss of <i>orf108</i>. Furthermore, fertility restorer genes of the two species were involved in the processing of the mRNA of the male sterility genes at different sites.

A cross-species approach for the identification of Drosophila male sterility genes

Male reproduction encompasses many essential cellular processes and interactions. As a focal point for these events, sperm offer opportunities for advancing our understanding of sexual reproduction at multiple levels during development. Using male sterility genes identified in human, mouse and fruit fly databases as a starting point, 103 Drosophila melanogaster genes were screened for their association with male sterility by tissue-specific RNAi knockdown and CRISPR/Cas9-mediated mutagenesis. This list included 56 genes associated with male infertility in the human databases, but not found in the Drosophila database, resulting in the discovery of 63 new genes associated with male fertility in Drosophila. The phenotypes identified were categorized into six distinct classes affecting sperm development. Interestingly, the second largest class (Class VI) caused sterility despite apparently normal testis and sperm morphology suggesting that these proteins may have functions in the mature sperm following spermatogenesis. We focused on one such gene, Rack 1, and found that it plays an important role in two developmental periods, in early germline cells or germline stem cells and in spermatogenic cells or sperm. Taken together, many genes are yet to be identified and their role in male reproduction, especially after ejaculation, remains to be elucidated in Drosophila, where a wealth of data from human and other model organisms would be useful.

Comparative cytological and transcriptome analysis reveals high pollen fertility and upregulation of environmentally sensitive genic male sterility genes in neo-tetraploid rice

Background: Autotetraploid rice is a useful germplasm for polyploid rice breeding; however, low seed setting is a major hindrance for the utilization of autotetraploid rice. Our previous study demonstrated that neo-tetraploid rice have great yield potential, which is thought to be one effective way to overcome the low fertility of autotetraploid rice. However, there is little known about the cause of high pollen fertility in neo-tetraploid rice. Here, we employed cytology and RNA-seq to study the molecular genetic mechanism of high pollen fertility in neo-tetraploid rice. Results: Cytological observations indicate that H1 displayed high pollen fertility (95.62%), lower percentage of pollen mother cells（PMCs）abnormalities, and stable chromosome configurations during the pollen development process compared with its two parents. RNA-seq analysis detected 440 differentially expressed genes (DEGs) in neo-tetraploid rice compared with its two parents. Of these DEGs, 193 were annotated as pollen fertility-related genes, and 129 (~66.8%) exhibited significant upregulation in neo-tetraploid rice compared with its two parents, including nine cloned genes ( TMS9-1 , TMS5 etc.) that were validated by qRT-PCR and had been demonstrated to be pollen fertility-related genes. We further selected TMS9-1 and TMS5 as the candidate gene and analysed its pollen fertility in neo-tetraploid rice using the CRISPR/Cas9 technique. Significant variations have been detected in pollen fertility value, pollen development process and expression level in H1 and its knock out lines. Conclusion: Our finding provides strong evidence for the regulatory mechanisms of neo-tetraploid rice, and upregulation of pollen fertility-related genes should be associated with high fertility. Moreover, knockout of environmentally sensitive genic male sterility genes in the present study provides the new useful germplasm for polyploidy rice breeding.

Comparative cytological and transcriptome analysis reveals high pollen fertility and upregulation of environmentally sensitive genic male sterility genes in neo-tetraploid rice

Background: Autotetraploid rice is a useful germplasm for polyploid rice breeding; however, low seed setting is a major hindrance for the utilization of autotetraploid rice. Our previous study demonstrated that neo-tetraploid rice have great yield potential, which is thought to be one effective way to overcome the low fertility of autotetraploid rice. However, there is little known about the cause of high pollen fertility in neo-tetraploid rice. Here, we employed cytology and RNA-seq to study the molecular genetic mechanism of high pollen fertility in neo-tetraploid rice. Results: Cytological observations indicate that H1 displayed high pollen fertility (95.62%), lower percentage of pollen mother cells（PMCs）abnormalities, and stable chromosome configurations during the pollen development process compared with its two parents. RNA-seq analysis detected 1479 differentially expressed genes (DEGs) in neo-tetraploid rice compared with its two parents. Of these DEGs, 433 were annotated as pollen fertility-related genes, and 240 (~55.4%) exhibited significant upregulation in neo-tetraploid rice compared with its two parents, including nine cloned genes ( TMS5 , CSA etc.) that were validated by qRT-PCR and had been demonstrated to be pollen fertility-related genes. We further selected TMS5 as a candidate gene and analysed its phenotype in neo-tetraploid rice using the CRISPR/Cas9 technique. Significant variations have been detected in phenotypic charts, pollen development process and expression level in H1 and TMS5 knockout lines. Conclusion: Our finding provides strong evidence for the regulatory mechanisms of neo-tetraploid rice, and upregulation of pollen fertility-related genes should be associated with high fertility. Moreover, knockout of environmentally sensitive genic male sterility genes in the present study provide the new useful germplasm for polyploidy rice breeding.

Independent domestications shape the genetic pattern of a reproductive isolation system in rice

Severe reproductive isolation (RI) exists between the two subspecies of rice, Indica and Japonica, but in the wild ancestors no post-zygotic RI was found. The studies about the establishment of the interspecies RI of rice are still rear. A pair of rice hybrid sterility genes, DOPPELGANGER 1 (DPL1) and DOPPELGANGER 2 (DPL2), offers a convenient example to study the evolutionary history of RI genes. Either of the two loci has one non-functional allele (DPL1- and DPL2-). The hybrid pollen carrying both DPL1- and DPL2- will be sterility.We collected 811 individuals: Oryza sativa (132), the two wild ancestors O. nivara (296) and O. rufipogon (383) as well as 20 DPL1 and 34 DPL2 sequences of O. sativa from on-line databases. We analysed the genetic and geographic pattern of DPLs in all three species to determine the origination regions of DPL1- and DPL2-. The neutral test as well as the diversities of nucleotide and haplotype were used to detect if selection shaped the pattern of DPLs.We found that DPL1- and DPL2- of rice emerged from wild ancestor populations in South Asia and South China through two respective domestications. Comparing with the ancestral populations, DPL1- and DPL2- both showed reduce of diversities, however their frequencies increased in rice. We assume that the reduce of diversities due to the bottleneck effect of domestication while the loss of one copy was preferred by artificial selection for cost savings.