Genetic Analysis and Mapping of Genes Involved in Fertility of Pingxiang Dominant Genic Male Sterile Rice

Pingxiang-dominant genic male sterile rice （PDGMSR） was the first dominant genic male sterile mutant identified in rice （Oryza sativa L.）, and the corresponding dominant genic male sterile gene was designated as Ms-p. The fertility of PDGMSR can be restored by introduction of a dominant epistatic fertility restoring gene in some rice varieties. In the present study, E823, an indica inbred rice variety, restored the fertility of PDGMSR, and the genetic pattern was found to be consistent with a dominant epistatic model, therefore, the dominant epistatic fertility restorer gene was designated as Rfe. The F2 population from the cross of PDGMSR/E823 was developed to map gene Rfe. The F2 plants with the genotypes Ms-pMs-pRferfe or Ms-pms-pRferfe were used to construct a fertile pool, and the corresponding sterile plants with genotypes Ms-pMs-prferfe or Ms-pms-prferfe were used to con- struct a sterile pool. The fertility restoring gene Rfe was mapped to one side of the microsatellite markers RM311 and RM3152 on rice chromosome 10, with genetic distances of 7.9 cM and 3.6 cM, respectively. The microsatellite markers around the location of the Ms-p gene were used to finely map the Ms-p gene. The findings of this study indicated that the microsatellite markers RM171 and RM6745 flanked the Ms-p gene, and the distances were 0.3 cM and 3.0 cM, respectively. On the basis of the sequence of rice chromosome 10, the physical distance between the two markers is approximately 730 kb. These findings facilitates molecular marker-assisted selection （MAS） of genes Ms-p and Rfe in rice breeding programs, and cloning them in the future.

Studies on Pingxiang dominant genic male sterile (PDGMS) rice

So far,14 examples of dominant genic malesterility on 10 crop species have been discov-ered in the world.In crossing sterile plants ofnon-pollen-type Fof cultivar-Wild combina-tlon(Ping＇ai 58/Huaye)as female parent withfertile plants Fof the wild-cultivar combination(Huaye/Ping＇ai 58)as male parent,a typ-ical abortive plant was found in the progeny.By excluding the Probability of balancedlethal,gametophyte sterility,and chromo-some aberration,studies showed that thesterility was controlled by a dominant malesterile gene in the nucleus.The material was

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.

Study on Seed Vigor of Rice Photo-ThermoSensitive Genic Male Sterile(PTGMS) Line with Low Temperature Storage

To explore the change rule of seed vigor and seed application technology of rice PTGMS lines with low temperature storage,Zhun S,Y58S,Feng S and Guangzhan 63-4S stored in the seed bank with the temperature of 8-10 ℃ and with the relative humidity of 55% for different years were used as the materials to study the properties of seed vigor,growth and development in the field, etc. The re- sults showed that the germination potential, germination rate, germination index and vigor index of rice PTGMS lines seeds showed downward trends as the time of low temperature storage prolonged,and all of the germination rates of the seeds within five years were over 60%. The conductivity in the soaked solution of seeds was significantly increased with the prolonged low temperature storage time. Seedling rate,root length,white root number,base stem width and seedling dry weight of seeds showed downward trends as the time of low temperature storage prolonged. There was no significant difference between two integral seedling qualities of seeds within 4-6 years in low temperature storage. Compared with the germination rate of low temperature storage seeds soaking for 6,12 and 24 h,the appropriate soak time of long time low temperature storage or low vigor seeds was 12 h. A serious natural aging phenomenon occurred in low temperature storage seeds after out of the storage. The germination rate of seeds placed under natural condition for 50 d decreased obviously. The duration from seeding to heading of Zhun S,Y58S and Feng S shortened as the time of low temperature storage prolonged, while the vari- ation of panicle duration of all PTGMS lines was not obvious. In conclusion, rice PTGMS line seeds with low temperature storage in 5 years could still be used nor- mally. However.seeds after low temperature storage should be sowed as soon as possible, and the soaking time of low vigor seeds should be shortened,and the possibility that the decreased duration from seeding to heading should also be con- sidered.

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.

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.

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.

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.

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.

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.

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.

Plant Temperature for Sterile Alteration of a Temperature-Sensitive Genic Male Sterile Rice,Peiai64S

The forecast of sterile alteration for the temperature-sensitive genic male sterile （TGMS） line in two-line hybrid rice seed production was traditionally based on screen temperature determined by weather station. The article put forward a new approach based on plant temperature, which was more exact and direct than the traditional method. The result of the simulation of the self-seeded setting rate of a widely used TGMS line, Peiai64S, by several temperature parameters and durations, showed that the fertility was directly affected by the plant temperature at a height of 20 cm or the air temperature around it in three days duration. Using the stem temperature of three days at a height of 20 cm as the simulation parameter, the fertility of Peiai64S had the maximum, minimum and optimum temperatures as 22.8, 21.7 and 22.5℃, respectively, whereas 23.2, 21.5 and 21.8℃ when using the air temperature of three days around the height of 20 cm as the parameter. Such temperature indices can be used to conclude the sterile alteration of TGMS for safeguarding seed production of twoline hybrid rice. The article also established a statistic model to conclude plant temperature by water temperatures at inflow and outflow, and air temperature and cloudage from weather station.

Genetic Mechanism of Dominant Earliness in Kefeng A,a New Rice Cytoplasmic Male Sterile Line

Kefeng A is an early maturing indica cytoplasmic male sterile （CMS） line of rice. Combinations derived from Kefeng A and late maturing indica restorer lines showed dominant earliness to various extents. To understand the genetic basis of dominant earliness, the genotype of photoperiod-sensitive genes in Kefeng A was analyzed using a complete set of heading time near isogenic lines （NILs） EGO to EG7, ER, LR, T65, T65E^b, T65E^bm, T65m, NIL（Hd1） and NIL（Hd4）. Results indicated that Kefeng A contained two dominant photoperiod-sensitive alleles E1 and Se-1^U on E1 and Se-1 loci, respectively, and the genotype of photoperiod-sensitivity genes for heading time in Kefeng A was E1E1e2e2E3E3Se-1^USe-1UEf-1Ef-1. Based on the detected heading time genotype, in combination with the heading time of Kefeng A and the early maturing phenomenon in its derived F1 hybrids, it is speculated that Kefeng A might carry a dominant inhibitor gene Su-E7 for the dominant photoperiod- sensitive gene E1, and a recessive inhibitor gene i-Se-1 for another dominant photoperiod-sensitive gene Se-1. The reason why F1 hybrids from Kefeng A exhibited early maturing was hereby analyzed and the breeding value of dominant earliness related genes in Kefeng A was discussed.

Progresses and Strategies of Gene Mapping and Isolating of Photo(thermo) sensitive Genic Male Sterile Rice

Considering the research on classical genetics of photoperiod(therm) sensitive genic male sterile rice, it is important to select the sterile lines and their segregating population controlled by one pair of gene in mapping and isolating sterile genes. It is discussed the advantages, disadvantages and the reasons leading to various mapping results of chromosome location of sterile genes through gene marker, isozyme marker and DNA marker techniques. In comparison to isolation of photo(thermo) sensitive sterile genes via various plant gene cloning techniques, it was concluded that map based cloning was acceptable, but it is still difficult to locate the loci of sterile genes within 1cM. On the other hand, “sensitivity to environment”, an important characteristic of sterile lines can be fully utilized by DD PCR and (or) RDA techniques. Therefore, these two techniques were considered as the effective ways to isolate sterile genes.

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.

Inducing fertility restoration in the genic male sterile line of rice with chemical regulators

For the first time, the fertility of rice genic male sterile line was partially restored with the application of chemical regulators at Hainan Rice Breeding Nursery on Mar 1993. A single panicle of the rice plant could bear as many as 27 grains. The chemical agent that could modulate the growth of the rice plants of genic male sterile line was developed by Prof ZHOU Guangqia and his colleagues at the Biology Institute, Hunan Teachers University, China.

Characterization of Reverse Thermo-sensitive Genic Male Sterile Lines in Cold Regions of China

Yannong S, a reverse thermo-sensitive genic male sterile （TGMS） line, is sterile at high temperature and fertile at low temperature. In the present study, the fertility of Yannong S and the japonica reverse TGMS lines derived from it was in- vestigated in Harbin, Heilongjiang from 2013 to 2015. The results showed that Yan- nong S was completely male sterile in July and August （the hottest months of the year in Harbin）, and the seed setting rate of bagged panicles was 0. We also in- ferred that the critical temperature for inducing the sterility of Yannong S was higher than 28 ℃. Most of the japonica reverse TGMS lines derived from Yannong S were completely aborted in July and August, with a seed setting rate of 0. It can be con- cluded that the reverse TGMS lines derived from Yannong S are genetically stable lines that have a long period of sterility in cold regions, so they may be the ideal genetic materials for the development and in-situ seed production of japonica hybrid rice.

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.

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.

A Co-Dominant Marker BoE332 Applied to Marker-Assisted Selection of Homozygous Male-Sterile Plants in Cabbage (Brassica oleracea var.capitata L.)

The dominant genic male sterility （DGMS） gene CDMs399-3 derived from a spontaneous mutation in the line 79-399-3 of spring cabbage （Brassica oleracea var. capitata L.）, has been successfully applied in hybrid seed production of several cabbage cultivars in China. During the development of dominant male sterility lines in cabbage, the conventional identification of homozygous male-sterile plants （CDMs399-3/CDMs399-3） is a laborious and time-consuming process. For marker-assisted selection （MAS） of the gene CDMs399-3 transferred into key spring cabbage line 397, expressed sequence tag-simple sequence repeats （EST-SSR） and SSR technology were used to identify markers that were linked to CDMs399-3 based on method of bulked segregant analysis （BSA）. By screening a set of 978 EST-SSRs and 395 SSRs, a marker BoE332 linked to the CDMs399-3 at a distance of 3.6 cM in the genetic background of cabbage line 397 were identified. 7 homozygons male-sterile plants in population P1170 with 20 plants were obtained finally via MAS of BoE332. Thus, BoE332 will greatly facilitate the transferring of the gene CDMs399-3 into the key spring cabbage line 397 and improve the application of DGMS in cabbage hybrid breeding.