Characterization and identification of the candidate gene of rice thermo-sensitive genic male sterile gene <Emphasis Type="Italic">tms5</Emphasis> by mapping

Previous research has demonstrated that the thermo-sensitive genic male-sterile (TGMS) gene in rice was regulated by temperature. TGMS rice is important to hybrid rice production because the application of the TGMS system in two-line breeding is cost-effective, simple, efficient and overcomes the limitations of the cytoplasmic male sterility (CMS) system. AnnongS is the first discovered and deeply studied TGMS rice line in China. Previous studies have suggested that AnnongS-1 and Y58S, two derivative TGMS lines of AnnongS, were both controlled by a single recessive gene named tms5, which was genetically mapped on chromosome 2. In the current study, three populations (AnnongS-1 × Nanjing11, Y58S × Q611, and Y58S × Guanghui122) were developed to investigate the tms5 gene molecular map. Analysis of recombination events of sterile samples, utilizing 125 probes covering the tms5 region, suggested that the tms5 gene was physically mapped to a 19 kb DNA fragment between two markers, 4039-1 and 4039-2, located on the BAC clone AP004039. Following the construction of a physical map between the two markers, ONAC023, a member of the NAC (NAM-ATAF-CUC-related) gene family, was identified as the candidate of the tms5 gene.     

Fine mapping of the rice thermo-sensitive genic male-sterile gene <Emphasis Type="Italic">tms5</Emphasis>

AnnongS-1, a thermo-sensitive genic male-sterile (TGMS) rice line, has a new TGMS gene. Genetic analysis indicated that the sterility of AnnongS-1 was controlled by a single resessive gene named tms5. In our previous studies based on an F₂ population from the cross between AnnongS-1 and Nanjing11, tms5 was mapped on chromosome 2. Recently, a RIL (recombinant inbred line) population from the same cross was developed and used for the fine mapping of the tms5 gene. Molecular marker techniques combined with BSA (bulked segregant analysis) were used. As a result, two AFLP markers (AF10, AF8), one RAPD marker (RA4), one STS marker (C365-1), one CAPs marker (G227-1) and four SSR markers (RM279, RM492, RM327, RM324) were found to be closely linked to tms5 gene. The DNA sequences of the RFLP marker of C365 and G227 were found in GenBank, and on the basis of these sequences, many primers were designed to amplify the two parents and their RIL population plants. Finally, the tms5 gene was mapped between STS marker C365-1 and CAPs marker G227-1 at a distance of 1.04 cM from C365-1 and 2.08 cM from G227-1.Communicated by H.F. LinskensY.G. Wang and Q.H. Xing contributed equally to this contribution.     

温度对双低两用核不育水稻96-5-2S与培矮64S育性的影响

在自然变温、人工控温及冷水灌溉条件下,比较研究了温度对双低两用核不育水稻96－5－2S与两用核不育水稻培矮64S育性影响的差异。结果表明：（1）当它们在雄性育性转换温敏感期1－12d平均自然日均温23.0-23.8℃的低温时,96－5－2S表现不良,套袋自交结实率为0,而培矮46S可育,套袋自交结实率为0．1％－4．5％;（2）在它们雄性育性转换温敏感期用22℃恒温处理5d,96－5－2S败育彻底,套袋自交结实率为0,而培矮64S可育,套袋自交结实率为10．7％;用17℃恒温处理6d,96－5－2S与培矮64S均可育,但96－5－2S套袋自交结实率（6．8％）显著高于培矮64S（2．5％）;（3）在它们雄性育性转换温和不同温度的冷水串灌15d,水深维持在20cm左右,当水温为22－22．5℃时,96－5－2S不育,结实率为0,而培矮64S可育,结实率为18．5％;当水温为19．5－21．5℃时,96－5－2S与培矮64S均可育,但96－5－2S结实率（2．5％－45．1％）显著或极显著低于培矮64S（50．4％－56．9％）以上结果说明：导致双低两用核不育水稻96－5－2S雄性不育的起点温度与导致其生理不育的下限温度均低,其不育性比培矮64S更稳定,耐寒性比培矮64S更强,即可确保制种安全,又可确保自身繁殖,对加快两系法杂交水稻的发展步伐将起到重要的促进作用。     

Molecular mapping of two reverse photoperiod-sensitive genic male sterility genes (rpms1 and rpms2) in rice (Oryza sativa L.)

The reverse photoperiod-sensitive genic male sterility (PGMS) and thermo-sensitive genic male sterility (TGMS) lines have an opposite phenotype compared with normal PGMS and TGMS lines widely used by the two-line system in current hybrid rice seed production. Thus, the application of reverse PGMS and TGMS lines can compensate PGMS and TGMS lines in hybrid rice production. YiD1S is a reverse PGMS line, in which pollen fertility is mainly regulated by day-length, but also influenced by temperature. Genetic analysis indicated that male sterility of YiD1S was controlled by two recessive major genes. An F₂ population from a cross between YiD1S and 8528 was developed and used for molecular mapping of the two reverse PGMS genes which were first named rpms1 and rpms2. Both simple sequence repeat (SSR) markers and bulked segregant analysis (BSA) were used in this study. As a result, one reverse PGMS gene (rpms1) was mapped to the interval between SSR markers RM22980 (0.9 cM) and RM23017 (1.8 cM) on chromosome 8. Eight SSR markers, YDS818, RM22984, RM22986, RM22997, YDS816, RM23002, RM339 and YDS810 completely co-segregated with the rpms1 gene. Another reverse PGMS gene (rpms2) was mapped to the interval between SSR markers RM23898 (0.9 cM) and YDS926 (0.9 cM) on chromosome 9. The physical mapping information from publicly available resources shows that the rpms1 and rpms2 loci are located in a region of 998 and 68 kb, respectively. The analysis based on marker genotypes showed that the effect of rpms1 was slightly larger than that of rpms2 and that the two genes interacted in controlling male sterility. H. F. Peng, Z. F. Zhang and B. Wu contributed equally to this work.     

Fine mapping and candidate gene analysis of the novel thermo-sensitive genic male sterility tms9-1 gene in rice

Key message

Fine mapping of the novel thermo-sensitive genic male sterility locus tms9 - 1 in the traditional TGMS line HengnongS-1 revealed that the MALE STERILITY1 homolog OsMS1 is the candidate gene.

Abstract

Photoperiod-thermo-sensitive genic male sterility (P/TGMS) has been widely used in the two-line hybrid rice breeding system. HengnongS-1 is one of the oldest TGMS lines and is often used in indica two-line breeding programs in China. In this study, our genetic analysis showed that the TGMS gene in HengnongS-1 was controlled by a single recessive gene that was non-allelic with the other TGMS loci identified, including C815S, Zhu1S and Y58S. Using SSR markers and bulked segregant analysis, we located the TGMS locus on chromosome 9 and named the gene tms9-1. Fine mapping further narrowed the tms9-1 loci to a 162 kb interval between two dCAPS markers. Sequence analysis revealed that a T to C substitution results in an amino acid change in the tms9-1 candidate gene (Os09g27620) in HengnongS-1 as compared to Minghui63. Sequencing of other rice accessions, including six P/TGMS lines, seven indica varieties and nine japonica varieties, showed that this SNP was exclusive to HengnongS-1. With multiple sequence alignment and expression pattern analyses, the rice MALE STERILITY1 homolog OsMS1 gene was identified as the candidate gene for tms9-1. Therefore, our study identified a novel TGMS locus and will facilitate the functional identification of the tms9-1 gene. Moreover, the markers linked to the tms9-1 gene will provide useful tools for the development of new TGMS lines by marker-assisted selection in two-line hybrid rice breeding programs.     

Low temperature compensates for defective tapetum initiation to restore the fertility of the novel TGMS line ostms15

In rice breeding, thermosensitive genic male sterility (TGMS) lines based on the tms5 locus have been extensively employed. Here, we reported a novel rice TGMS line ostms15 (Oryza sativa ssp. japonica ZH11) which show male sterility under high temperature and fertility under low temperature. Field evaluation from 2018 to 2021 revealed that its sterility under high temperature is more stable than that of tms5 (ZH11), even with occasional low temperature periods, indicating its considerable value for rice breeding. OsTMS15 encodes an LRR-RLK protein MULTIPLE SPOROCYTE1 (MSP1) which was reported to interact with its ligand to initiate tapetum development for pollen formation. In ostms15, a point mutation from GTA (Val) to GAA (Glu) in its TIR motif of the LRR region led to the TGMS phenotype. Cellular observation and gene expression analysis showed that the tapetum is still present in ostms15, while its function was substantially impaired under high temperature. However, its tapetum function was restored under low temperature. The interaction between mOsTMS15 and its ligand was reduced while this interaction was partially restored under low temperature. Slow development was reported to be a general mechanism of P/TGMS fertility restoration. We propose that the recovered protein interaction together with slow development under low temperature compensates for the defective tapetum initiation, which further restores ostms15 fertility. We used base editing to create a number of TGMS lines with different base substitutions based on the OsTMS15 locus. This work may also facilitate the mechanistic investigation and breeding of other crops.     

Genetic characterization and fine mapping of a novel thermo-sensitive genic male-sterile gene tms6 in rice (Oryza sativa L.)

The application of genetic male sterility in hybrid rice production has great potential to revolutionize hybrid seed production methodology. The two-line breeding system by using thermo-sensitive genic male sterility (TGMS) has been discovered and successfully developed as a breeding strategy in rice. One TGMS gene was investigated by a spontaneous rice mutant line, Sokcho-MS, originated from a Korean japonica variety. It was shown that Sokcho-MS is completely sterile at a temperature higher than 27°C and/or lower than 25°C during the development of spikelets, but fertile at the temperature ranging from 25 to 27°C regardless of the levels of day-length. Genetic analysis and molecular mapping based on SSR, STS and EST markers revealed that a single recessive gene locus involved the control of genic male sterility in Sokcho-MS. By using an F₂ mapping population derived from a cross between Sokcho-MS and a fertile indica variety Neda, the new TGMS gene, designated as tms6, was mapped primarily to the long arm of chromosome 5 of Oryza sativa at the interval between markers E60663 (2.0 cM) and RM440 (5.8 cM). Subsequently, tms6 was fine mapped to the interval between markers RM3351 (0.1 cM) and E60663 (1.9 cM). As tms6 appeared to be independent of other mapped TGMS genes in rice, the genetic basis of Sokcho-MS was further discussed.     

Effect of Growth Regulators and Chemicals on Pollen Sterility in TGMS Lines of Rice

Thermosensitive genic male sterility (TGMS) in rice is a widely adopted technique for successful hybrid rice production in Asia. TGMS lines remain male sterile when daily mean temperature is above the critical sterility temperature and are therefore used as female parents. The same line will remain fertile when mean temperature is below the critical sterility temperature. Achievement of 100% male sterility in TGMS lines is important for the successful utilization of TGMS lines as female parents in hybrid rice production. This study examined the external application of some growth regulators and chemicals and their effect on pollen sterility. Among the various treatments, ethrel (800 ppm), salicylic acid (600 ppm) and maleic hydrazide (0.2%) induced a significantly higher percentage of male sterility in the TGMS lines. The sprayed plants also showed higher total phenol accumulation in their flag leaves. The results suggest that it is possible to achieve 100% male sterility in TGMS lines with the external application of growth regulators and chemicals.     

Photoperiod- and thermo-sensitive genic male sterility in rice are caused by a point mutation in a novel noncoding RNA that produces a small RNA

Photoperiod- and thermo-sensitive genic male sterility (PGMS and TGMS) are the core components for hybrid breeding in crops. Hybrid rice based on the two-line system using PGMS and TGMS lines has been successfully developed and applied widely in agriculture. However, the molecular mechanism underlying the control of PGMS and TGMS remains obscure. In this study, we mapped and cloned a major locus, p/tms12-1 (photo- or thermo-sensitive genic male sterility locus on chromosome 12), which confers PGMS in the japonica rice line Nongken 58S (NK58S) and TGMS in the indica rice line Peiai 64S (PA64S, derived from NK58S). A 2.4-kb DNA fragment containing the wild-type allele P/TMS12-1 was able to restore the pollen fertility of NK58S and PA64S plants in genetic complementation. P/TMS12-1 encodes a unique noncoding RNA, which produces a 21-nucleotide small RNA that we named osa-smR5864w. A substitution of C-to-G in p/tms12-1, the only polymorphism relative to P/TMS12-1, is present in the mutant small RNA, namely osa-smR5864m. Furthermore, overexpression of a 375-bp sequence of P/TMS12-1 in transgenic NK58S and PA64S plants also produced osa-smR5864w and restored pollen fertility. The small RNA was expressed preferentially in young panicles, but its expression was not markedly affected by different day lengths or temperatures. Our results reveal that the point mutation in p/tms12-1, which probably leads to a loss-of-function for osa-smR5864m, constitutes a common cause for PGMS and TGMS in the japonica and indica lines, respectively. Our findings thus suggest that this noncoding small RNA gene is an important regulator of male development controlled by cross-talk between the genetic networks and environmental conditions.     

Identification and fine mapping of rtms1-D,a gene responsible for reverse thermosensitive genic male sterility from Diannong S-1X

Thermosensitive genic male sterility (TGMS) has been widely used in two-line hybrid rice breeding. Due to hybrid seed production being highly affected by changeable environments, its application scope is limited to some extent. Thus, it is of great importance to identify potential TGMS genes in specific rice varieties. Here, Diannong S-1 xuan (DNS-1X), a reverse TGMS (RTGMS) japonica male sterile line, was identified from Diannong S-1. Genetic analysis showed that male sterility was tightly controlled by a single recessive gene, which was supported by the phenotype of the F₁ and F_2:3 populations derived from the cross between DNS-1X and Yunjing 26 (YJ26). Combining simple sequence repeat (SSR) markers and bulked segregation analysis (BSA), we identified a 215 kb region on chromosome 10 as a candidate reverse TGMS region, which was designated as rtms1-D. It was narrower than the previously reported RTGMS genes rtms1 and tms6(t). The fertility conversion detected in the natural environment showed that DNS-1X was sterile below 28–30 °C; otherwise, it was fertile. Histological analysis further indicated that the pollen abortion was occurred in the young microspore stage. This study will provide new resources for two-line hybrid rice and pave the way for molecular breeding of RTGMS lines.     

Comparative proteomics analysis reveals the mechanism of fertility alternation of thermosensitive genic male sterile rice lines under low temperature inducement

Thermosensitive genic male sterile (TGMS) rice line has made great economical contributions in rice production. However, the fertility of TGMS rice line during hybrid seed production is frequently influenced by low temperature, thus leading to its fertility/sterility alteration and hybrid seed production failure. To understand the mechanism of fertility alternation under low temperature inducement, the extracted proteins from young panicles of two TGMS rice lines at the fertility alternation sensitivity stage were analyzed by 2DE. Eighty‐three protein spots were found to be significantly changed in abundance, and identified by MALDI‐TOF‐TOF MS. The identified proteins were involved in 16 metabolic pathways and cellular processes. The young panicles of TGMS rice line Zhu 1S possessed the lower ROS‐scavenging, indole‐3‐acetic acid level, soluble protein, and sugar contents as well as the faster anther wall disintegration than those of TGMS rice line Zhun S. All these major differences might result in that the former is more stable in fertility than the latter. Based on the majority of the 83 identified proteins, together with microstructural, physiological, and biochemical results, a possible fertile alteration mechanism in the young panicles of TGMS rice line under low temperature inducement was proposed. Such a result will help us in breeding TGMS rice lines and production of hybrid seed.     

Molecular mapping of a rice gene conditioning thermosensitive genic male sterility using AFLP, RFLP and SSR techniques

The discovery and application of the thermosensitive genic male sterility (TGMS) system has great potential for revolutionizing hybrid seed production technology in rice. Use of the TGMS system in two-line breeding is simple, inexpensive, efficient, and eliminates the limitations associated with the cytoplasmic-genetic male sterility (CMS) system. An F₂ population developed from a cross between a TGMS indica mutant, TGMS–VN1, and a fertile indica line, CH1, was used to identify molecular markers linked to the TGMS gene and to subsequently determine its chromosomal location on the linkage map of rice. Bulk segregant analysis was performed using the AFLP technique. From the survey of 200 AFLP primer combinations, four AFLP markers (E2/M5–600, E3/M16–400, E5/M12–600, and E5/M12–200) linked to the TGMS gene were identified. All the markers were linked to the gene in the coupling phase. All except E2/M5–200 were found to be low-copy sequences. However, the marker E5/M12–600 showed polymorphism in RFLP analysis and was closely linked to the TGMS gene at a distance of 3.3 cM. This marker was subsequently mapped on chromosome 2 using doubled-haploid mapping populations derived from the crosses IR64×Azucena and CT9993×IR62666, available at IRRI, Philippines, and Texas Tech University, respectively. Linkage of microsatellite marker RM27 with the TGMS gene further confirmed its location on chromosome 2. The closest marker, E5/M12–600, was sequenced so that a PCR marker can be developed for the marker-assisted transfer of this gene to different genetic backgrounds. The new TGMS gene is tentatively designated as tms4(t). Received: 13 July 1999 / Accepted: 27 July 1999     

Strategy and utilization of a herbicide resistance gene in two-line hybrid rice

Hybrid rice plays an important role in China's aim to improve rice production as it accounts for some 50% of rice planting area but produces about 60% of the total rice grain. However, the existing three-line system used in hybrid rice production has its limitations. The two-line system, which makes use of photoperiod-sensitive genic male-sterile (PGMS) and thermo-sensitive genic male-sterile (TGMS) lines to generate the male-sterile parental line, was developed to overcome some of these limitations. The sterility of the male-sterile line of two-line hybrid rice, however, fluctuates when the temperature-sensitive phase of fertility encounters abnormal low temperatures during hybrid seed production, which induces selfing and decreases the purity of hybrid. We describe here the strategy of utilizing a herbicide resistance gene in two-line hybrid rice to eliminate this fluctuation in the sterility of the P/TGMS lines during hybrid seed production and reports the development of the herbicide resistance restorer line Bar68-1 and its herbicide-resistant early season hybrid rice Xiang125s/Bar68-1. When the restorer line and its derived hybrid are herbicide resistant, the selfed seeds can be removed easily from the hybrid by herbicide spraying. A herbicide resistance gene bar was transferred into a restorer line by particle bombardment. The resulting transgenic restorer line Bar68-1 and its hybrid Xiang125 s/Bar68-1 inherited stable herbicide resistance. The purity of Xiang125s/Bar68-1 was increased by spraying the seed bed with herbicide, which resulted in a significant increase in yield, grain quality, and disease resistance in comparison to the controls in a regional trial.     

Marker-assisted breeding of a photoperiod-sensitive male sterile <Emphasis Type="Italic">japonica</Emphasis> rice with high cross-compatibility with <Emphasis Type="Italic">indica</Emphasis> rice

The incomplete fertility of japonica × indica rice hybrids has inhibited breeders’ access to the substantial heterotic potential of these hybrids. As hybrid sterility is caused by an allelic interaction at a small number of loci, it is possible to overcome it by simple introgression at the major sterility loci. Here we report the use of marker-assisted backcrossing to transfer into the elite japonica cv. Zhendao88 a photoperiod-sensitive male sterility gene from cv. Lunhui422S (indica) and the yellow leaf gene from line Yellow249 (indica). The microsatellite markers RM276, RM455, RM141 and RM185 were used to tag the fertility genes S5, S8, S7 and S9, respectively. Line 509S is a true-breeding photoperiod-sensitive male sterile plant, which morphologically closely resembles the japonica type. Genotypic analysis showed that the genome of line 509S comprises about 92% japonica DNA. Nevertheless, hybrids between line 509S and japonica varieties suffer from a level of hybrid sterility, although the line is highly cross-compatible with indica types, with the resulting hybrids expressing a significant degree of heterosis. Together, these results suggest that segment substitution on fertility loci based on known information and marker-assisted selection are an effective approach for utilizing the heterosis of rice inter-subspecies.     

A bentazon and sulfonylurea sensitive mutant: breeding,genetics and potential application in seed production of hybrid rice

The use of a thermosensitive genic male sterility (TGMS) system in two-line hybrid rice breeding is affected greatly by the sterility instability of TGMS lines caused by temperature fluctuation beyond their critical temperatures for fertility reversion. To prevent seed production from self contamination, we have developed a system to secure seed purity using a herbicide-sensitive TGMS mutant, M8077S, obtained by radiation. Genetic analysis, using the F₁, F₂ and F₃ populations derived from this mutant and other normal varieties, revealed that bentazon lethality/sensitivity was controlled by a single recessive gene, which was named bel. The mutant can be killed at the seedling stage by bentazon at 300 mg/l or higher, a dosage that is safe for its F₁ hybrids and all other normal varieties. This mutant is also sensitive to all the tested sulfonylurea herbicides. Response of segregating plants to these two types of herbicide indicated that sulfonylurea sensitivity was also controlled by bel. By crossing this mutant with Pei-Ai 64S, an F₂ population was developed for genetic mapping. Surveying the two DNA pools from sensitive and non-sensitive F₂ plants identified four markers that were polymorphic between the pools. The putative linked markers were then confirmed with the F₂ population. The bel locus was located on chromosome 3, 7.1 cM from the closest microsatellite marker RM168. Phenotypic analysis indicated that the bel gene had no negative effect on agronomic traits in either a homozygous or heterozygous status. The mutant M8077S is valuable in the development of a TGMS breeding system for preventing impurity resulting from temperature fluctuation of the TGMS. Several two-line hybrid rice crosses using this system are under development.     

Identification of a deletion in <Emphasis Type="Italic">tms2</Emphasis> and development of gene-based markers for selection

Rice is one of the most important food crops. The temperature-sensitive genic male sterility (TGMS) system provides a great potential for improving food production by hybrids. The use of TGMS system is simple, inexpensive, effective, and eliminates the limitations of the conventional three-line system. A rice gene, tms2, generated by irradiation of a japonica variety has been reported to control TGMS in several rice lines. Previous studies reported genetic markers linked to this gene, and the gene was transferred to an aromatic Thai cultivar. Using information obtained from published databases, we located positions of the reported genetic markers flanking the gene in rice genomic sequences, and developed gene-based markers located inside the flanking markers for polymorphism detection. We found that inbred indica tms2 mutant plants contain about 1 Mb of japonica DNA, in which at least 70 kb was deleted. Using RT-PCR for expression analysis, four genes out of seven genes annotated as expressed proteins located inside the deletion showed expression in panicles. These genes could be responsible for TGMS phenotypes of tms2. In addition, we developed gene-based markers flanking and inside the deletion for selecting the tms2 gene in breeding populations. By genotyping 102 diverse rice lines including 38 Thai rice lines, 5 species of wild rice, and 59 exotic rice lines including TGMS lines and cultivars with desirable traits, a gene-based marker located inside the deletion and one flanking marker were shown to be highly specific for the tms2 mutant.     

Developing disease‐resistant thermosensitive male sterile rice by multiplex gene editing

High-quality and disease-resistant male sterile lines have great potential for applications in hybrid rice breeding. We introduced specific mutations into the TMS5, Pi21, and Xa13 genes in Pinzhan intermediate breeding material using the CRISPR/Cas9 multiplex genome editing system. We found that the transgenefree homozygous triple tms5/pi21/xa13 mutants obtained in the T1 generation displayed characteristics of thermosensitive genic male sterility(TGMS) with enhancedresistance to rice blast and bacterial blight. Our study provides a convenient and effective way of converting breeding intermediate material into TGMS lines through multiplex gene editing, which could significantly accelerate the breeding of sterile lines.