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.     

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     

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.     

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 of a gene for non-pollen type thermosensitive genic male sterility in rice (Oryza sativa L.)

The thermo-sensitive genic male sterility (TGMS) lines play a crucial role in two-line hybrid rice production. For a practical TGMS line, the stability of male sterility is one of the most important technical indicators. In this study, XianS, a spontaneous mutant with stable male sterility from an indica rice cultivar Xianhuangzhan, was classified as a non-pollen type TGMS line. The critical non-pollen sterility point temperature of XianS was determined as 27°C. Genetic analysis demonstrated that the non-pollen sterility in XianS was controlled by a single recessive gene. Using SSR markers and bulked segregant analysis, the TGMS gene in XianS was fine mapped to a 183 kb interval between RMAN81 and RMX21 on chromosome 2. Two markers, 4039-1 and RMX14 completely cosegregated with this gene. Allelism test indicated that the non-pollen phenotype in seven non-pollen type TGMS lines from different sources, XianS, AnnongS-1, Q523S, Q524S, N28S, G421S, and Q527S is caused by the same TGMS gene. Although the location of TGMS gene in XianS is close to the gene OsNAC6, a previously identified candidate gene of tms5 in AnnongS-1, the sequence of OsNAC6 and its promoter region was identical in TGMS line XianS, AnnongS-1, and wild-type Xianhuangzhan. These results suggest that the non-pollen type TGMS trait probably be controlled by the same TGMS gene in different TGMS rice lines, but its real candidate gene still need to be further studied and identified.     

Molecular mapping of a thermosensitive genetic male sterility gene in rice using bulked segregant analysis.

The thermosensitive genetic male sterility (TGMS) system is considered to be a more efficient alternative to the cytoplasmic male sterility (CMS) system for hybrid rice. An F2 population from a cross between a TGMS mutant line (IR32364TGMS) and IR68 was used to map the TGMS gene tms3(t). Fertile and sterile bulks were constructed following the classification of F2 plants into true breeding sterile, fertile, and segregating fertile plants based on F3 family studies. From the survey of 389 arbitrary primers in bulked segregant analysis, four RAPD markers were identified in which three, OPF182600, OPB19750, and OPAA7550, were linked to tms3(t) in repulsion phase and one, OPAC3640, was linked to tms3(t) in coupling phase. The tms3(t) gene was flanked by OPF182600 and OPAC3640 on one side and by OPAA7550 and OPB19750 on the other side. All four markers were low-copy sequences and two of them (OPF182600 and OPAC3640) detected polymorphism when the markers were used to probe the genomic blots. Subsequently, OPAC3640 was mapped to the short arm of chromosome 6 using a mapping population available at IRRI. However, no RFLP markers from this region showed linkage to tms3(t) owing to the lack of polymorphism between the parents. All RAPD fragments were cloned and partially sequenced from both ends. Thus, PCR primers can be designed to develop PCR markers for marker-assisted breeding to facilitate the transfer of tms3(t) from one genetic background to another.     

Genetic analyses and mapping of a new thermo-sensitive genic male sterile gene in maize

The present study describes a novel thermo-sensitive genic male sterile (TGMS) line, Qiong68ms. To analyse the mode of fertility inheritance and tag the TGMS gene, a set of F₂, BC₁ and F_2:3 populations derived from a cross between Qiong68ms and K12 were evaluated for a period of 2 years. Classical genetic analyses and QTL mapping using the mean restoration percentage of the F_2:3 populations revealed that the fertility of Qiong68ms was likely to be governed by a single recessive gene, which was named tms3; the tms3 gene was mapped to a location between SSR markers umc2129 and umc1041, at a distance of 3.7 cM form umc2129 and 1.5 cM form umc1041. The molecular markers tightly linked with tms3 gene will aid in the transfer of the TGMS gene to various background inbred lines using the MAS method.     

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.     

Development of PCR-based markers for thermosensitive genetic male sterility gene tms3(t) in rice (Oryza sativa L.)

Development of simple and reliable PCR-based markers is an important component of marker-aided selection (MAS) activities for agronomically important genes in rice breeding. In order to develop PCR-based markers for a rice thermosensitive genetic male sterility gene tms3(t), located on chromosome 6, the nucleotide sequences of four linked RAPD markers OPF18(2600), OPAC3(640), OPB19(750) and OPM7(550) were used to design and synthesize several pairs of specific primers for PCR amplification of the genomic DNA of both the parents IR32364TGMS (sterile) and IR68 (fertile), involved in mapping this gene. For the RAPD marker OPF 18(2600), two pairs of specific primer pair combination from different positions of the sequence resulted in generation of two codominant STS (Sequence Tagged Sites) markers. In case of markers OPAC3(640), OPB19(750) and OPAA7(550) the first two could generate dominant polymorphism, while the last one could not be successful in PCR amplification. Both the codominant STSs with primer combinations F18F/F18RM and F18FM/F18RM were found to be tightly linked to the tms3(t) gene with a genetic distance of 2.7 cM. The sizes of the different alleles in case of F18F/F18RM, F18FM/F18RM combinations were 2300 bp, 1050 bp, and 1900 bp, 1000 bp respectively. The efficiency of marker-assisted selection for this trait was estimated as 84.6%. Polymorphism survey of 12 elite rice lines, indicated that these PCR-based markers for tms3(t) can now be used in selecting TGMS plants at seeding stage in the segregating populations in environment independent of controlled temperature regime.     

Molecular mapping of the reverse thermo-sensitive genic male-sterile gene (rtms1) in rice

TGMS (thermo-sensitive genic male-sterile) rice is widely used in hybrid rice production. Because of a specific temperature requirement, it can be used only in a narrow rice-growing zone in Asia. A newly discovered reverse thermo-sensitive genic male-sterile line, J207S, has an opposite phynotype compared to the normal TGMS lines. J207S is completely sterile when the temperature is lower than 31°C. Thus, it can be widely used in a larger area. Genetic analysis indicated that the sterility of J207S was controlled by a single recessive gene which was first named as rtms1. An F₂ population from the cross between J207S and E921 was developed and used for molecular mapping of the rtms1 gene. The AFLP (amplified fragment length polymorphism) technique, combined with BSA (bulked segregant analysis), was used to screen markers linked to the target gene, and eight polymorphic AFLP loci were identified. Co-segregating analysis using the F₂ population showed that two of them, Rev1 and Rev7, were closely linked to the target gene with a recombinant rate of 3.8% and 7.7%, respectively. Both Rev1 and Rev7 were found to be single-copy sequences through Southern analysis. Rev1 was subsequently mapped on chromosome 10 with a doubled-haploid mapping populations derived from the cross CT9993 × IR62266 available at Texas Tech University. RM222 and RG257 were linked to Rev1 at a distance of 11.8 cM and 4.6 cM, respectively. Additional SSR markers from the rice map of Cornell University, RFLP markers from the map of RGP in Japan and the map of Texas Tech University were selected from the region surrounding Rev1 on chromosome 10 to conduct the fine-mapping of the rtms1 gene. Presently, rtms1 was mapped between RM239 and RG257 with genetic distance of 3.6 cM and 4.0 cM, respectively. The most-closely linked AFLP marker, Rev1, 4.2 cM from the rtms1 gene, was sequenced and converted into a SCAR (sequence characterized amplified region) marker which could facilitate marker-assisted selection of the rtms1 gene. Received: 2 November 2000 / Accepted: 21 November 2000     

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.     

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.     

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.     

Aanlysis of short photo-periodic sensitive genic male sterility and molecular mapping of rpms3(t) gene in rice (Oryza sativa L.) using SSR markers

A research was conducted on the pollen fertility of rice sterile lines D52S and D38S responsive to photoperiod during the sensitive stage under natural and controlled conditions. Bulk segregant analysis (BSA) and recessive class approach were applied to identify DNA markers that co-segregate with gene conferring male-sterility in D52S mutant rice. The results showed that in day-light higher or equal to 14.00 h, D52S and D38S rice pollen were fertile; however, they were sterile when day-length was less than 14.00 h. They were therefore considered to be short photo-periodic sensitive genic male sterile lines(Short PGMS lines). Under short day-light conditions, the pollen fertility segregation of F₂ populations from crosses between D52S/Shuhui527 and D52S/Gui99showed 3:1 ratio of fertile to sterile plants suggestingthat male sterility in D52S was controlled by one recessive gene. Two markers RM244 and RM216 located on chromosome number 10 co-segregated completely with the rpms locus. The locus was mapped to the interval between SSR markers RM2571 (6.6 cM) and RM244 (4.6 cM).     

萍乡显性核不育水稻育性相关基因的定位和遗传分析

萍乡显性核不育水稻(Pingxiang Dominant Genic Male Sterile Rice,PDGMSR)是在水稻中首次发现的显性核不育材料,其育性由两对显性基因互作控制,一对是萍乡显性核不育基因Ms-p,另一对是显性上位恢复基因(dominant epistatic fertility restorer gene,Rfe)。两者共同存在时显性上位恢复基因能抑制不育基因的表达,从而使育性表现可育。本实验用一个对萍乡显性核不育水稻有恢复能力的水稻品种E823与萍乡显性核不育水稻配制杂交组合,将(萍乡核不育水稻/E823)F2作为定位群体,根据F3株系的育性分离,选择育性分离株系对应F2单株(基因型为Ms-pMs-pRefrfe和Ms-pms-pRferfe)构建可育池,用对应F2株系中的不育单株(基因型为Ms-pMs-prferfe或Ms-pms-prferfe)构建不育池,将显性上位恢复基因Rfe定位在水稻10染色体RM311和RM3152一侧,遗传距离分别为7.9cM和3.6cM。根据已有的Ms-p的定位结果,合成10染色体部分微卫星引物,对不育单株进行分析,发现RM171和RM6745位于Ms-p的两侧,距离分别为0.3cM和3.0cM。根据10染色体的测序结果,将Ms-p界定在约730kb的范围内,并构建了Ms-p的电子重叠群。植物显性核不育的育性恢复机理存在“复等位基因”和“显性上位互作”两种假说,贺浩华等用经典的遗传学方法证明了萍乡显性核不育水稻育性恢复的遗传机理属于“显性上位互作”。理论上认为,确定其遗传机理最为有效的方法是基因定位,如果不育基因和恢复基因位于同一位点,则其遗传机理属于“复等位基因”,否则为“显性上位互作”。本实验将不育基因和恢复基因定位在水稻10染色体不同的位点,用基因定位的方法证实了萍乡显性核不育水稻育性恢复的遗传机理属于“显性上位互作”。     

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.     

Fine mapping and candidate gene analysis of <Emphasis Type="Italic">ptgms2-1</Emphasis>, the photoperiod-thermo-sensitive genic male sterile gene in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Photoperiod-thermo-sensitive genic male sterile (PTGMS) rice exhibits a number of desirable traits for hybrid rice production. The cloning genes responsible for PTGMS and those elucidating male sterility mechanisms and reversibility to fertility would be of great significance to provide a foundation to develop new male sterile lines. Guangzhan63S, a PTGMS line, is one of the most widely used indica two-line hybrid rice breeding systems in China. In this study, genetic analysis based on F₂ and BC₁F₂ populations derived from a cross between Guangzhan63S and 1587, determined a single recessive gene controls male sterility in Guangzhan63S. Molecular marker techniques combined with bulked-segregant analysis (BSA) were used and located the target gene (named ptgms2-1) between two SSR markers RM12521 and RM12823. Fine mapping of the ptgms2-1 locus was conducted with 45 new Insertion–Deletion (InDel) markers developed between the RM12521 and RM12823 region, using 634 sterile individuals from F₂ and BC₁F₂ populations. Ptgms2-1 was further mapped to a 50.4 kb DNA fragment between two InDel markers, S2-40 and S2-44, with genetic distances of 0.08 and 0.16 cM, respectively, which cosegregated with S2-43 located on the AP004039 BAC clone. Ten genes were identified in this region based on annotation results from the RiceGAAS system. A nuclear ribonuclease Z gene was identified as the candidate for the ptgms2-1 gene. This result will facilitate cloning the ptgms2-1 gene. The tightly linked markers for the ptgms2-1 gene locus will further provide a useful tool for marker-assisted selection of this gene in rice breeding programs.     

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.     

Genetic analysis of temperature-sensitive male sterilty in rice

The present study of genetic analysis is an attempt to precisely characterize diverse temperature-sensitive genic male-sterile (TGMS) lines so as to explore the possibilities of utilizing the most promising in large-scale hybrid seed production. Genetical studies revealed that the TGMS segregants derived from crosses involving TGMS lines ID24 and SA2 expressed differential fertility levels at low-temperature conditions. A majority of these progenies expressed transgressive segregation towards either sterility of fertility, causing instability of sterility and low reversibilty of fertility which may be due to large numbers of single-locus QTLs and their epistatic interactions. We identified two putative genes imparting temperature-sensitive male sterility after observing crosses involving diverse TGMS sources. To identify suitable molecular markers closely linked to the trait we used RAPD, AFLP and microsatellites which generated polymorphism through bulked segregant analysis. AFLP analysis using a smaller genome kit resulted in enormous polymorphism, out of which the combination EAA/MCAG amplified a 330-bp fragment, which closely segregated with the gene at a distance of 5.3 cM. This fragment was eluted for cloning and from the sequence a STS primer (TS200) was developed which produced a dominant polymorphism specific to TGMS. The microsatellite RM257, located earlier on chromosome 9, was linked with the TGMS trait in SA2 at a distance of 6.2 cM. RM257 produced a codominant polymorphism with 145-bp (sterile) and 132-bp (fertile) products. Both individually and collectively, the markers TS200 and RM257 located on either side of the TGMS locus are very useful for marker-assisted selection. Received: 10 April 1999 / Accepted: 29 July 1999     

Fine mapping of the recessive genic male sterility gene (<Emphasis Type="Italic">Bnms3</Emphasis>) in <Emphasis Type="Italic">Brassica napus</Emphasis> L.

The Brassica napus oilseed rape line, 7-7365AB, is a recessive epistatic genic male sterile (RGMS) two-type line system. The sterility is controlled by two pairs of recessive duplicate genes (Bnms3 and Bnms4) and one pair of recessive epistatic inhibitor gene (Bnrf). Homozygosity at the Bnrf locus (Bnrfrf) inhibits the expression of the two recessive male sterility genes in homozygous Bnms3ms3ms4ms4 plants and produces a male fertile phenotype. This line has a good potential for heterosis utilization but it is difficult to breed heterotic hybrids without molecular markers. To develop markers linked to the BnMs3 gene, amplified fragment length polymorphism (AFLP) technology was applied to screen the bulks of sterile and fertile individuals selected randomly from a population of near-isogenic lines (NIL) consisting of 2,000 plants. From a survey of 1,024 primer combinations, we identified 17 AFLP markers linked to the BnMs3 gene. By integrating the previous markers linked to the BnMs3 gene into the genetic map of the NIL population, two markers, EA01MC12 and EA09P06, were located on either side of the BnMs3 gene at a distance of 0.1 and 0.3 cM, respectively. In order to use the markers for male sterile line breeding, five AFLP markers, P05MG05, P03MG04, P11MG02, P05MC11₂₅₀, and EA09P06, were successfully converted into sequence characterized amplified region (SCAR) markers. Two of these, P06MG04 and sR12384, were subsequently mapped on to linkage group N19 using two doubled-haploid mapping populations available at our laboratory derived from the crosses Tapidor × Ningyou7 and Quantum × No2127-17. The markers found in the present study should improve our knowledge of recessive genic male sterility (RGMS), and accelerate the development of male sterile line breeding and map-based cloning.