Anther culture in rice proportionally rescues microspores according to gametophytic gene effect and enhances genetic study of hybrid sterility

BackgroundTo investigate plant hybrid sterility, we studied interspecific hybrids of two cultivated rice species, Asian rice (Oryza sativa) and African rice (O. glaberrima). Male gametes of these hybrids display complete sterility owing to a dozen of hybrid sterility loci, termed HS loci, but this complicated genetic system remains poorly understood.ResultsMicrospores from these interspecific hybrids form sterile pollen but are viable at the immature stage. Application of the anther culture (AC) method caused these immature microspores to induce callus. The segregation distortion of 11 among 13 known HS loci was assessed in the callus population. Using many individual calli, fine mapping of the HS loci was attempted based on heterozygotes produced from chromosome segment substitution lines (CSSLs). Transmission ratio distortion (TRD) from microspores was detected at 6 of 11 HS loci in the callus population. The fine mapping of S-1 and S-19 loci using CSSLs revealed precise distances of markers from the positions of HS loci exhibiting excessive TRD.ConclusionsWe demonstrated that AC to generate callus populations derived from immature microspores is a useful methodology for genetic study. The callus population facilitated detection of TRD at multiple HS loci and dramatically shortened the process for mapping hybrid sterility genes

Genetic Mapping of the Gamete Eliminator Locus, S-2, Causing Hybrid Sterility and Transmission Ratio Distortion Found between Oryza sativa and Oryza glaberrima Cross Combination

Hybrid sterility is a reproductive barrier that prevents gene flow between species. In Oryza species, some hybrid sterility loci, which are classified as gamete eliminators, cause pollen and seed sterility and sex-independent transmission ratio distortion (siTRD) in hybrids. However, the molecular basis of siTRD has not been fully characterized because of lacking information on causative genes. Here, we analyze one of the hybrid sterility loci, S-2, which was reported more than forty years ago but has not been located on rice chromosomes. Hybrids between African rice (Oryza glaberrima) and a near-isogenic line that possesses introgressed chromosomal segments from Asian rice (Oryza sativa) showed sterility and siTRD, which confirms the presence of the S-2 locus. Genome-wide SNP marker survey revealed that the near-isogenic line has an introgression on chromosome 4. Further substitution mapping located the S-2 locus between 22.60 Mb and 23.54 Mb on this chromosome. Significant TRD in this chromosomal region was also observed in a calli population derived from cultured anther in hybrids of another cross combination of African and Asian rice species. This indicates that the pollen abortion caused by the S-2 locus occurs before callus induction in anther culture. It also suggests the wide existence of the S-2-mediated siTRD in this interspecific cross combination. Chromosomal location of the S-2 locus will be valuable for identifying causative genes and for understanding of the molecular basis of siTRD

Effectiveness of Create ML in microscopy image classifications: a simple and inexpensive deep learning pipeline for non-data scientists

Observing chromosomes is a time-consuming and labor-intensive process, and chromosomes have been analyzed manually for many years. In the last decade, automated acquisition systems for microscopic images have advanced dramatically due to advances in their controlling computer systems, and nowadays, it is possible to automatically acquire sets of tiling-images consisting of large number, more than 1000, of images from large areas of specimens. However, there has been no simple and inexpensive system to efficiently select images containing mitotic cells among these images. In this paper, a classification system of chromosomal images by deep learning artificial intelligence (AI) that can be easily handled by non-data scientists was applied. With this system, models suitable for our own samples could be easily built on a Macintosh computer with Create ML. As examples, models constructed by learning using chromosome images derived from various plant species were able to classify images containing mitotic cells among samples from plant species not used for learning in addition to samples from the species used. The system also worked for cells in tissue sections and tetrads. Since this system is inexpensive and can be easily trained via deep learning using scientists’ own samples, it can be used not only for chromosomal image analysis but also for analysis of other biology-related images

Diploid Male Gametes Circumvent Hybrid Sterility Between Asian and African Rice Species

In F-1 hybrids of Oryza sativa (Asian rice) and Oryza glaberrima (African rice), heterozygosity leads to a complete gamete abortion because of allelic conflict at each of the 13 hybrid sterility (HS) loci. We systematically produced 19 plants from the F-1 hybrids of both the rice species by the anther culture (AC) method. Five of the 19 interspecific hybrid plants were partially fertile and able to produce seeds. Unlike ordinal doubled haploid plants resulting from AC, these regenerated plants showed various ploidy levels (diploid to pentaploid) and different zygosities (completely homozygous, completely heterozygous, and a combination). These properties were attributable to meiotic anomalies in the interspecific hybrid F-1 plants. Examination of the genetic structures of the regenerated plants suggested meiotic non-reduction took place in the interspecific hybrid F-1 plants. The centromeric regions in the regenerated plants revealed that the abnormal first and/or second divisions of meiosis, namely the first division restitution (FDR) and/or second division restitution (SDR), had occurred in the interspecific hybrid. Immunohistochemical observations also verified these phenomena. FDR and SDR occurrences at meiosis might strongly lead to the formation of diploid microspores. The results demonstrated that meiotic anomalies functioned as a reproductive barrier occurred before the HS genes acted in gamete of the interspecific hybrid. Although such meiotic anomalies are detrimental to pollen development, the early rescue of microspores carrying the diploid gamete resulted in the fertile regenerated plants. The five partially fertile plants carrying tetraploid genomes with heterozygous alleles of the HS loci produced fertile diploid pollens, implying that the diploid gametes circumvented the allelic conflicts at the HS loci. We also proposed how diploid male gametes avoid HS with the killer-protector model

Diploid Male Gametes Circumvent Hybrid Sterility Between Asian and African Rice Species

In F_1 hybrids of Oryza sativa (Asian rice) and Oryza glaberrima (African rice), heterozygosity leads to a complete gamete abortion because of allelic conflict at each of the 13 hybrid sterility (HS) loci. We systematically produced 19 plants from the F1 hybrids of both the rice species by the anther culture (AC) method. Five of the 19 interspecific hybrid plants were partially fertile and able to produce seeds. Unlike ordinal doubled haploid plants resulting from AC, these regenerated plants showed various ploidy levels (diploid to pentaploid) and different zygosities (completely homozygous, completely heterozygous, and a combination). These properties were attributable to meiotic anomalies in the interspecific hybrid F_1 plants. Examination of the genetic structures of the regenerated plants suggested meiotic non-reduction took place in the interspecific hybrid F1 plants. The centromeric regions in the regenerated plants revealed that the abnormal first and/or second divisions of meiosis, namely the first division restitution (FDR) and/or second division restitution (SDR), had occurred in the interspecific hybrid. Immunohistochemical observations also verified these phenomena. FDR and SDR occurrences at meiosis might strongly lead to the formation of diploid microspores. The results demonstrated that meiotic anomalies functioned as a reproductive barrier occurred before the HS genes acted in gamete of the interspecific hybrid. Although such meiotic anomalies are detrimental to pollen development, the early rescue of microspores carrying the diploid gamete resulted in the fertile regenerated plants. The five partially fertile plants carrying tetraploid genomes with heterozygous alleles of the HS loci produced fertile diploid pollens, implying that the diploid gametes circumvented the allelic conflicts at the HS loci. We also proposed how diploid male gametes avoid HS with the killer–protector model