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.

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.

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

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.

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.

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.

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.

Differentially expressed miRNAs in anthers may contribute to the fertility of a novel Brassica napus genic male sterile line CN12A

In Brassica napus L.(rapeseed),complete genic male sterility(GMS)plays an important role in the utilization of heterosis.Although microRNAs(miRNAs)play essential regulatory roles during bud development,knowledge of how GMS is regulated by miRNAs in rapeseed is rather limited.In this study,we obtained a novel recessive GMS system,CN12AB.The sterile line CN12A has defects in tapetal diferentiation and degradation.llumina sequencing was employed to examine the expression of miRNAs in the buds of CN12A and the fertile line CN12B.We identifed 85 known miRNAs and 120 novel miRNAs that were expressed during rapeseed anther development.When comparing the expression levels of miRNAs between CN12A and CN12B,19 and 18 known miRNAs were found to be difrentially expressed in 0.5--1.0 mm buds and in 2.5--3.0 mm buds,respectively.Among these,the expression levels of 14 miRNAs were higher and the levels of 23 miRNAs were lower in CN12A compared with CN12B.The predicted target genes of these iferentially expressed miRNAS encode protein kinases,F-box domain-containing proteins,MADS-box family proteins,SBP-box gene family members,HD ZIP proteins,floral homeotic protein APETAL A 2(AP2)。and nuclear factor Y,subunit A.These targets have previously been reported to be involved in pollen development and male sterility,suggesting that miRNAs might act as regulators of GMS in rapeseed anthers.Furthermore,RT-qPCR data suggest that one of the dffrentially expressed miRNAs,bna-miR159,plays a role in tapetal differentiation by regulating the expression of transcription factor BnMYB101 and participates in tapetal degradation and infuences callose degradation by manipulating the expression of BnA6.These findings contibute to our understanding of the roles of miRNAs during anther development and the occurrence of GMS in rapeseed.

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.

Identification of Differentially Expressed Genes During Anther Abortion of Taigu Genic Male Sterile Wheat by Combining Suppression Subtractive Hybridization and cDNA Array

Talgu Genlc Male Sterile Wheat （TGMSW; Trltlcum aestlvum L.）, a dominant genic male sterile germplasm, is of considerable value in the genetic Improvement of wheat because of Its stable Inherence, complete male abortion, and high cross-fertilization rate. To Identify specially transcribed genes In sterile anther, a suppression subtractlve hybridization （aSH） library was constructed with sterile anther as the tester and fertile anther as the driver. A total of 2 304 SSH Inserts amplified by polymerase chain reaction were arrayed using robotic printing. The cDNA arrays were hybridized with 32P-labeled probes prepared from the RNA of forward- and reverse-subtracted anthers. Ninety-six clones were scored as upregulated in sterile anthers compared with the corresponding fertile anthers and some clones were selected for sequencing and analysis In GenBank. Based on their putative functions, 87 non-redundant clones were classified Into the following groups： （i） eight genes Involved In metabolic processes; （11） four material transportation genes; （iii） three signal transductlon-assoclated genes; （iv） four stress response and senescence-associated protein genes; （v） seven other functional protein genes; （vi） five genes with no known function; and （vii） another 56 genes with no match to the databases. To test the hybridization efficiency, eight genes were selected and analyzed by Northern blot. The results of the present study provide a comprehensive overview of the genes and gene products Involved In anther abortion In TGMSW.

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.

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.

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.

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.