Introgression of Gene for Non-Pollen Type Thermo-Sensitive Genic Male Sterility to Thai Rice Cultivars

For the two-line hybrid rice system, pol en sterility is regulated by recessive gene that responds to temperature. The recessive gene controlling thermo-sensitive genetic male sterility （TGMS） is expressed when the plants are grown in conditions with higher or lower critical temperatures. To transfer tgms gene（s） control ing TGMS to Thai rice cultivars by backcross breeding method, a male sterile line was used as a donor parent while Thai rice cultivars ChaiNat 1, PathumThani 1, and SuphanBuri 1 were used as recurrent parents. The BC2F2 lines were developed from backcrossing and selfing. Moreover, the simple sequence repeat （SSR） markers were developed for identifying tgms gene and the linked marker was used for assisting selection in backcrossing. The identification lines were confirmed by pol en observation. The results showed the success of introgression of the tgms gene into Thai rice cultivars. These lines will be tested for combining ability and used as female parent in hybrid rice production in Thailand.

Mechanism of Sterility and Breeding Strategies for Photoperiod/Thermo-Sensitive Genic Male Sterile Rice

To understand the male sterility mechanism of photoperiod/thermo-sensitive genic male sterile [P（T）GMS] lines in rice, the research progress on genetics of photoperiod and/or temperature sensitive genic male sterility in rice was reviewed. A new idea was proposed to explain the sterility mechanism of P（T）GMS rice. The fertility transition from sterile to fertile is the result of cooperative regulation of major-effect sterile genes with photoperiod and/or temperature sensitive genes, but not the so-called pgms gene in P（T）GMS rice. The minor-effect genes, which exhibit accumulative effect on sterility, are the important factors for the critical temperature of sterility transition. The more minor-effect genes the sterile line holds, the lower the critical temperature of sterility transition is. The critical temperature of sterility transition will be invariable if all the minor-effect genes are homozygous. The strategies for breeding P（T）GMS rice were also proposed. The selective indices of critical photoperiod and temperature for sterility transition should be set according to varietal type and ecological region. Imposing selection pressure is a key technology for breeding P（T）GMS rice with lower critical temperature for sterility, and improving the comprehensive performance of the whole traits and combining ability is vital for breeding P（T）GMS rice lines.

Plant Temperature and Its Simulation Model of Thermo-Sensitive Genic Male Sterile Rice

Plant temperature （Tp） and its relations to the microclimate of rice colony and irrigation water were studied using a thermo-sensitive genic male sterile （TGMS） rice line, Pei＇ai 64S. Significant differences in the daily change of temperature were detected between Tp and air temperature at the height of 150 cm （TA）. From 8：00 to 20：00, Tp was lower than TA, but they were similar during 21：00 to next 7：00. The maximum Tp occurred one hour earlier than the maximum TA, though they both reached the minimum at 6：00. Tp fluctuated less than TA. At the same height, during 6：00-13：00, Tp was higher than air temperature （Ta）, and Tp reached the maximum one hour earlier than Ta. During the rest time on sunny day, Tp was close to or even a little lower than Ta. On overcast day, Tp was higher than Ta in the whole day, and both maximized at the same time. In addition, Tp was regulated by solar radiation, cloudage and wind speed in daytime, and by irrigation water at night. The present study indicated that a TA of 29.6℃ was the critical point, at which Tp was increased or decreased by irrigation water. Tp and the difference between water and air temperatures showed a conic relation. Tp fluctuation was also regulated by the absorption or reflection of solar radiation by leaves during daytime and release of heat energy during nighttime. By analysis on correlation and regression simulation, two models of Tp were established.

Marker-Assisted Breeding of Thermo-Sensitive Genic Male Sterile Line 1892S for Disease Resistance and Submergence Tolerance

Rice line 1892S is an elite thermo-sensitive genic male sterile(TGMS)line for two-line hybrid rice production.However,1892S is susceptible to rice blast,bacterial blight and submergence.Here we reported the introduction of blast resistance(R)gene Pi9,bacterial blight R gene Xa21 and submergence tolerance gene Sub1A into 1892S genetic background through backcrossing and marker-assisted selection.The improved TGMS line 31892S and its hybrids conferred disease resistance to rice blast and bacterial blight,and showed submergence tolerance for over 14 d without significant loss of viability.The sterility-fertility conversion of 31892S was similar to that of 1892S.31892S and its derived hybrid rice had similar agronomic traits and grain quality with 1892S and the control hybrid rice,respectively.The newly developed 31892S provided an improved TGMS line for two-line hybrid rice production with disease resistance to rice blast and bacterial blight,and submergence tolerance with no yield penalty or change in grain quality.

Elongation of the Uppermost Internode for Changxuan 3S,a Thermo-Sensitive Genic Male Sterile Rice Line

Changxuan 3S, a thermo-sensitive genic male sterile （TGMS） rice line with eui gene, is derived from the TGMS rice line Pei＇ai 64S by irradiation with 350 Gy of ^60Co γ-ray. To elucidate the uppermost internode elongation of the TGMS line with eui gene, Changxuan 3S and its parent Pei＇ai 64S were used to study the effects of temperature on panicle exsertion. At 24℃, the uppermost internode of Changxuan 3S elongated the fastest from the 4^th day before flowering to 0 day （flowering）, being 2.1-fold as that of Pei＇ai 64S, whereas it elongated slowly during the 12^th day to the 4^th day before flowering and the 1^st to the 3^rd day after flowering. The uppermost internode of Changxuan 3S exserted from the flag leaf sheath at 22℃, 24℃ and 26℃, and the length of elongated uppermost internode increased with the decreasing temperatures. At 28℃, though the panicles of Changxuan 3S were still enclosed in the leaf sheath, the degree of panicle enclosure was significantly lower compared with Pei＇ai 64S. Cytological studies on Changxuan 3S showed that the uppermost internode elongation was attributed to the increase of cell number and cell elongation, and the latter was more significant. Moreover, the numbers of outermost and innermost parenchyma cells and the cell length of the uppermost internode reduced with the increasing temperatures.

Characterizations and identification of the candidate gene of rice thermo-sensitive genic male sterile gene tms5 by mapping

Previous study indicated that the thermo-sensitive genic malesterile(TGMS) gene in rice was regulated by temperature.TGMS rice plays an important role in hybrid rice production,because the application of the TGMS system in two-line breeding is laborsaving,timesaving,simple,inexpensive,efficient,and eliminating the limitations of the cytoplasmic male sterility(CMS) system.'AnnongS' is the first discovered and deeply studied TGMS rice lines in China.'AnnongS-1' and 'Y58S',two derivatives of TGMS line AnnongS,were both controlled by a single recessive gene named tms5,which was genetically mapped on chromosome 2.In this study,three populations('AnnongS-1' × 'Nanjing11','Y58S' × 'Q611',and 'Y58S' × 'Guanghui122') were developed and used for the molecular fine mapping of the tms5 gene.By analyzing recombination events in the sterile individuals using a total of 125 probes covering the tms5 region,the tms5 gene was physically mapped to a 19-kb DNA fragment between two markers 4039-1 and 4039-2,which were located on the BAC clone AP004039.After the construction of the physical map between two markers 4039-1 and 4039-2,a member(ONAC023) of the NAC(NAM-ATAF-CUC-related) gene family was identified as the candidate gene of the tms5 gene.

Characterization and fine mapping of RTMS10, a semi-dominant reverse thermo-sensitive genic male sterile locus in rice

The discovery and application of environment-sensitive genic male sterile(EGMS) rice germplasm provide an easy method for hybrid rice breeding and have made great contributions to hybrid rice production. Typically, the photoperiod-and thermosensitive GMS(P/TGMS) lines utilized in two-line hybrid systems are male sterile under long day or/and high temperature but fertile under short day or/and low temperature conditions. However, Yannong S(Yn S), a reverse TGMS(rTGMS) line, is sterile under low temperature(<29℃) and fertile under high temperature(>29.5℃). Here, we report a genetic study on the rTGMS trait in Yn S. Interestingly, the F1 plants of the cross between Yn S and a cultivar, L422, were male sterile at 22℃ and completely fertile at 27℃. Moreover, the segregation ratio of fertile and sterile individuals in Yn S/L422 F2 populations changed from 1:3.05 to 2.95:1 when the ambient temperature increased, showing that the rTGMS trait exhibits semidominance in Yn S. We further found a locus on chromosome 10, termed RTMS10, which controls the rTGMS trait in Yn S. We then finely mapped RTMS10 to a ~68 kb interval between markers ID13116 and ID1318 by Yn S/L422 BC6 F2 populations. A near iso-genic line(NIL) NL1 from the BC6 F3 generation was developed and the pollen of NL1 became abnormal from the meiosis stage under low temperature. In summary, we identified an rTGMS locus, RTMS10, and provided co-segregated markers, which could help to accelerate molecular breeding of rTGMS lines and better understand the rTGMS trait in rice.

Molecular Markers and Candidate Genes for Thermo-Sensitive Genic Male Sterile in Rice

The discovery of thermo-sensitive genic male sterility(TGMS) has led to development of a simple and highly efficient two-line breeding system. In this study, genetic analysis was conducted using three F_2 populations derived from crosses between IR68301 S, an indica TGMS rice line, and IR14632(tropical japonica), Supanburi 91062(indica) and IR67966-188-2-2-1(tropical japonica), respectively.Approximately 1:3 ratio between sterile and normal pollen of F_2 plants from the three populations revealed that TGMS is controlled by a single recessive gene. Bulked segregant analysis using simple sequence repeat(SSR) and insertion-deletion(InDel) markers were used to identify markers linked to the tms gene. The linkage analysis based on the three populations indicated that the tms locus was located on chromosome 2 covering the same area. Using IR68301S × IR14632 F_2 population, the results showed that the tms locus was located between SSR marker RM12676 and InDel marker 2gAP0050058. The genetic distance from the tms gene to these two flanking markers were 1.10 and 0.82 cM, respectively.InDel marker 2gAP004045 located between these two markers showed complete co-segregation with the TGMS phenotype. In addition, InDel marker vf0206114052 showed 2.94 cM linked to the tms gene using F_2 populations of IR68301S × Supanburi 91062. These markers are useful tool for developing new TGMS lines by marker-assisted selection. There were ten genes located between the two flanking markers RM12676 and 2gAP0050058. Using quantitative real-time PCR for expression analysis, 7 of the 10 genes showed expression in panicles, and response to temperatures. These genes could be the candidate gene controlling TGMS in IR68301S.

Improving Blast Resistance of a Thermo-Sensitive Genic Male Sterile Rice Line GD-8S by Molecular Marker-Assisted Selection

The broad-spectrum blast resistance gene Pi-1, from donor line BL122, was introduced into a thermo-sensitive genic male sterile rice line GD-8S, which possessed good grain quality but high susceptibility to rice blast, by using backcross breeding and molecular marker-assisted selection. Five elite improved male sterile lines, RGD8S-1, RGD8S-2, RGD8S-3, RGD8S-4 and RGD8S-5, were selected based on the results of molecular marker analysis, spikelet sterility, recovery rate of genetic background and agronomic traits. Thirty-three representative blast isolates collected from Guangdong Province, China were used to inoculate the improved lines and the original line GD-8S artificially. The resistance frequencies of the improved lines ranged from 76.47% to 100%, much higher than that of the original line GD-8S （9.09%）. On the agronomic characters, there were no significant differences between the improved lines and GD-8S except for flag leaf length and panicle number per plant. The improved lines could be used for breeding hybrid rice with high blast resistance.

Inheritance of the Male Sterility in a New Photo/Thermo-Sensitive Genie Male Sterile Line B06S of Rice

The major male sterile genes in a new photo/thermo-sensitive genie male sterile (PTGMS) line B06S of rice were analyzed by the manipulation of mixture distribution theory. The results indicated that a pair of major male sterile nuclear genes with large effects were responsible for controlling the male sterility of B06S.

Thermo-sensitive genic male sterile (TGMS) rice line developed by population improvement

In this study population improvement or random polycross plus mass selection was adopted with using N8S, Huaizao4, Xiang2B and Zaoyou1 as parents. To help comprehensively improve the important characters such as combining ability, grain quality, resistance, stigma exsertion rate, etc. The stigma exsertion rate of ZhunS, an elite TGMS line, is 78.6%, 72.4% higher than that of V20A. Hybrids produced by crossing ZhunS with R402, Minhui77 and Minhui63 increased the yield by 3.8%～5.8% and decreased grain chalkiness by 82.1%～84.4% compared with those from crossing V20A with the corresponding R lines.

Genetic Analysis and Primary Mapping of pms4, a Photoperiod-Sensitive Genic Male Sterility Gene in Rice (Oryza sativa)

To understand the genetic characteristics of a new photoperiod-sensitive genic male sterile line Mian 9S, some reciprocal crosses were made between Mian 9S and six indica rice materials, Yangdao 6, Luhui 602, Shuihui 527, Mianhui 725, Fuhui 838 and Yixiang 1B. Genetic analysis results suggested that the photoperiod-sensitive genic male sterility （PGMS） of Mian 9S was controlled by a single recessive nuclear gene. Thus, the F2 population derived from the cross of Yangdao 6/Mian 9S was used to map the PGMS gene in Mian 9S. By using SSR markers, the PGMS gene of Mian 9S was mapped on one side of the markers, RM6659 and RM1305, on rice chromosome 4, with the genetic distances of 3.0 cM and 3.5 cM, respectively. The gene was a novel PGMS gene and designated tentatively as pms4. In addition, the application of the pms4 gene was discussed.

Identification of a locus associated with genic male sterility in maize via EMS mutagenesis and bulked-segregant RNA-seq

Genic male sterility(GMS) is one of the most important resources for exploiting heterosis in crop breeding, so that identifying genomic loci regulating GMS is desirable. However, many regulatory genes controlling GMS have not yet been characterized in maize, owing partly to a lack of genetic materials. We generated a recessive male-sterile maize mutant in the Jing 724 genetic background via ethyl methanesulfonate treatment, and found the male sterility to be due to a single gene mutation. Bulk-segregant RNA sequencing of three replicates indicated that one genomic region located at the end of chromosome 4 was associated with the observed mutant phenotype. Among genes with nonsynonymous mutations,Zm00001 d053895(bHLH51) showed abolished expression in the sterile bulks and was annotated as a bHLH transcription factor orthologous to Arabidopsis AMS, suggesting an association with the male sterility of the mutant. Kompetitive Allele-Specific PCR assays further validated the exclusive correlation of male sterility with the single C-to-T mutation in the fifth exon. The new maize mutant and the potential SNP locus provide novel genetic material for investigating the molecular mechanism underlying tapetal development and may facilitate the improvement of hybrid production systems.

Development of Japonica Male Sterile Lines Integrating Cytoplasmic Male Sterility and Photosensitive Genic Male Sterility

Acknowledgement It has been previously established that the BT type of cytoplasmic male sterility （CMS） is induced by high temperatures, while photosensitive genic male sterility （PGMS） seed sets by low temperatures induce. In the current study, we have bred photosensitive cytoplasmic male sterility （PCMS） lines （2308SA and 2310SA） by crossing the CMS line with the PGMS japonica line with maintainer genes. The sterility of PCMS japonica was consequently controlled by two groups of male sterile genes resulting from the integration of PGMS and CMS genes. The results on plant fertility, at different sowing times, were as follows： （a） Under conditions of natural long-day photoperiod and at temperatures above 35~C, the PGMS gene regulated PCMS japonica sterility - the higher the temperature, the lower the pollen fertility. However, bagged seed sets of PCMS japonica, not exposed to high temperatures, induced the CMS seed set. （b） Exposure to long-day photoperiod and temperature conditions between 35℃ and the critical sterility inducing temperature of PGMS resulted in both PGMS and CMS gene controlled sterility of PCMS japonica, which exhibited stable characteristics. （c） When exposed to critical sterility inducing temperatures or short-day photoperiod and daily high temperatures below 32℃, the BT type of the CMS gene regulated PCMS sterility. Under these conditions, the PGMS gene rendered male sterility insusceptible to occasional cool summer days when this PCMS line, adopted for hybrid seed production, develops into panicle differentiation stage. The present study also investigated the fertility restoration, seed production and combining ability of PCMS japonica so as to optimize its use.

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-1 X),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 F;and F;populations derived from the cross between DNS-1 X 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 rtmsl-D.It was narrower than the previously reported RTGMS genes rtmsl and tms6(t).The fertility conversion detected in the natural environment showed that DNS-1 X was sterile below 28-30℃;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.

Breeding double low super hybrids with dominant genic male sterility three lines in Brassica napus

A double low homozygous two-type sterile line ZY-2AB, its near-isogenic temporary maintainer line ZY-4, and the full sterile line ZY-3 derived from the cross of ZY-2A with ZY-4, were developed to breed three-line dominant GMS hybrid in Brassica napus. Three years data showed that the ratios of sterile and fertile plants fit the expected 1:1 ratio in ZY-2AB, and the 1:0 ratio in ZY-3, respectively. Some double low super hybrid combinations were screened out when the full sterile line ZY-3 was used as female parent, among which the combination 01Z24 increased 36.88 percent more yield than control and the combination 98Z131 has been passed through the Sichuan Provincial Regional Test and registered in Sichuan Province. Two years data showed that the agronomic performance of the two-line hybrids derived from crosses of ZY-2A with restorer lines versus that of the three-line hybrids produced by ZY-3 was not different for any of the nine most important agronomic characteristics, revealing that near-isogenic line of homozygous two-type line was used as temporary maintainer could effectively avoid the disadvantage of three-way cross hybrid in breeding three-line dominant GMS hybrid.

Mapping of a gene for photoperiod-sensitive genic male sterility in Nongken 58s on chromosome 5 of rice

A photoperiod-sensitive genic male sterile (PGMS) rice was found in 1973 as a spontaneous mutant of Nongken 58, a japonica variety. Pollen fertility of Nongken 58s (N58s) is completely sterile when grown under long-day conditions, whereas fertile under short-day conditions. This PGMS was found to be controlled by one or two recessive gene(s), of which one gene(pms)was linked to a marker gene(d-1) on chromosome 5. In order to identify a more precise location of the pms, we analyzed the populations of BCFand BCFof N58s//N58s/KL211(v-10, virescent) and N58s//N58s/KL520 (gh-1, gold hull). The marker genes v-10 and gh-1 are located on the flanking region of d-1. The F, plants of two crosses were fertile. The number of fertile and sterile individuals in BCFfit

Isolation and Analysis of the Promoter of OsRacD from PhotoperiodSensitive Genic Male Sterile Rice

By using OsRacD cDNA as probe to screen the genomic library of photoperiod sensitive genic male sterile rice line Nongken 58S, a positive clone containing 2 kb promoter and 396 bp coding region of OsRacD was obtained. Compared with the promoter of OsRacD cloned by reverse PCR from normal rice variety Nongken 58 （Nongken 58N）, the homology was 99.8%, and the different nucleotides were outside the predicted response elements in promoter, suggesting that the fertility between rice varieties Nongken 58S and Nongken 58N under the long-day conditions was not attributed to the difference in the structure of OsRacD upstream regulation sequences, but to the developmental regulation of gene differential expression.

New Temperature Sensitive Genic Male Sterile Lines with Better Outcrossing Ability for Production of Two-Line Hybrid Rice

An investigation was carried out with three newly developed temperature sensitive genic male sterile （TGMS） lines for their floral traits, seed production potential and outcrossing ability in ten cross combinations. In the TGMS lines, fertile pollens had an average diameter of 0.89 mm while the sterile pollens was with 0.02 mm diameter.TS-29-150GY produced the biggest fertile pollens with 0.92 mm and other two lines produced relatively smaller pollens with 0.91 and 0.85 ram. Pollen fertility during the fertility reversion period was an average of 60.7%. TS-29-150GY had the maximum of 66.9% spikelet fertility whereas other two lines （TNAU18S and TNAU60S） had relatively lower spikelet fertility of 27.8% and 26.7%, respectively. Average of 17.00 g of seed yield was obtained in the TGMS lines during the fertility reversion period. TS-29- 150GY had the highest value of 21.20 g of seed yield while TNAU18S and TNAU60S produced 16.6 g and 13.2 g of seed yield, respectively. The low seed production ability of these three TGMS lines was attributed only to the environmental conditions prevailing during the period. All three TGMS lines had considerable outcrossing potential of 41.2%, 24.6% and 25.0%, respectively. The cross combinations viz. TNAU18S/IET21508 （36 g/plant）, TNAU18S/IET21044 （13 g/plant）, TNAU18S/IET21009 （26.5 g/plant）, TNAU60S/CB-09-106 （26.2 g/plant）, TNAU60S/IET21009 （14 g/plant） and TS29-150-GY/DRR 3306 （39.2 g/plant） showed perfect synchronization with acceptable hybrid seed yield, indicating suitability of TGMS system under Indian condition. Based on the outcrossing related traits viz. panicle exertion, angle of glume opening, stigma length and pollen size, TNAU18S was identified as the best, followed by TS-29-150GY.