Thermo-sensitive Genic Male Sterile Line Research Articles

Elongation of the uppermost internode for Shuangdi Pei eS, TGMS rice with eui gene

Shuangdi Pei eS, a thermo-sensitive genic male sterile (TGMS) rice line with eui gene is derived from TGMS rice line Shuangdi S by irradiation with 350 Gy60Co γ-ray. To elucidate the uppermost internode elongation of TGMS line with eui gene, Shuangdi Pei eS and its original TGMS line Shuangdi S were used to study the effects of temperature on panicle exsertion. At 25°C, the uppermost internode of Shuangdi Pei eS had the quickest elongation from the 4th day before flowering to 0 day (flowering), being 2.1-fold as that of Shuangdi S, whereas it elongated slowly during the 12th day to the 4th day before flowering and the 1st to the 3rd day after flowering. The uppermost internode of Shuangdi Pei eS exserted from the flag leaf sheath at 23, 25 and 27°C, respectively, and the length of elongated uppermost internode increased with the decreasing temperatures. At 30°C, though the panicles of Shuangdi Pei eS were still enclosed in the leaf sheath, the degree of panicle enclosure was significantly lower compared with Shuangdi S. Cytological studies on Shuangdi Pei eS which 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. Key words: Rice (Oryza sativa L.), thermo-sensitive genic male sterility (TGMS), elongated uppermost internode gene (eui), panicle exsertion, temperature.

Developing thermo-sensitive genic male sterile lines in rice

The three-line method (A/B/R) of heterosis breeding is cumbersome and warrants development of alternate approaches to exploit hybrid vigor commercially in rice. Two-line breeding is one such possibility that emerged following the chance discovery of photoperiod-sensitive genic male sterile (PGMS) lines and thermosensitive genic male sterile (TGMS) lines in China.

Male sterility and fertility behavior of suspected thermosensitive genic male sterile (TGMS) lines

The male sterility-fertility response in thermosensitive genic male sterile (TGMS) lines was studied to identify their adaptability to different temperature regimes. Twenty suspected TGMS lines available at the Paddy Breeding Station, Coimbatore, were used for the study. The plants of these lines, which were completely male sterile during summer 1995, were tagged and observed for their male sterility-fertility behavior in the ratooned tillers during winter 1995 and summer 1996 by staining pollen grains with I-KI. Panicle development stages, from the formation of pollen mother cells to the late uninucleate stage of pollen grains, were considered as sensitive to temperature. These stages occurred 9-19 d prior to heading (or 9-17 d after panicle initiation). Therefore, the critical temperature at which plants showed variation in pollen sterility and fertility was calculated by considering the average daily temperature and average maximum temperature prevalent during 9-19 d prior to heading.

Field evaluation of thermosensitive genic male sterile lines

The use of thermosensitive genic male sterility (TGMS) is well recognized in two-line heterosis breeding. The use of the TGMS system has several advantages over the cytoplasmic genic male sterility system, such as (1) its simple and efficient seed production system, and (2) its greater scope for enhancing heterosis because any genotype can be used as a male parent. To develop hybrids using the TGMS system, it is necessary to identify or develop lines that show a desirable transformation from fertility to sterility and vice versa.

Genetics of the thermosensitive genic male sterility trait in rice

UPRI 95-140 (P1), the thermosensitive genic male sterile (TGMS) line of low temperature fertility transformation type, was analyzed for its inheritance and transformation behavior. Parents, F1, F2, and six test crosses between TGMS (female parent) and UPRI 95-141 (P2), UPRI 95-165 (P3), UPRI 95-124 (P4), UPRI 95-117 (P5), and IR36 (P6) were screened in the field during the main season.

Identifying rice genotypes with the TGMS trait suitable for north India

The discovery of the environment-sensitive genic male sterility (EGMS) system laid the foundation for replacing the three-line system with the simpler and more efficient two-line system for hybrid rice seed production. EGMS includes photoperiod-sensitive genic male sterility (PGMS) and thermosensitive genic male sterility (TGMS) systems. The required day length differences do not occur in our rice seasons for PGMS seed production. Therefore, we attempted to develop suitable TGMS lines in India.

Uncovering Small RNA-Mediated Responses to Cold Stress in a Wheat Thermosensitive Genic Male-Sterile Line by Deep Sequencing

The male sterility of thermosensitive genic male sterile (TGMS) lines of wheat (Triticum aestivum) is strictly controlled by temperature. The early phase of anther development is especially susceptible to cold stress. MicroRNAs (miRNAs) play an important role in plant development and in responses to environmental stress. In this study, deep sequencing of small RNA (smRNA) libraries obtained from spike tissues of the TGMS line under cold and control conditions identified a total of 78 unique miRNA sequences from 30 families and trans-acting small interfering RNAs (tasiRNAs) derived from two TAS3 genes. To identify smRNA targets in the wheat TGMS line, we applied the degradome sequencing method, which globally and directly identifies the remnants of smRNA-directed target cleavage. We identified 26 targets of 16 miRNA families and three targets of tasiRNAs. Comparing smRNA sequencing data sets and TaqMan quantitative polymerase chain reaction results, we identified six miRNAs and one tasiRNA (tasiRNA-ARF [for Auxin-Responsive Factor]) as cold stress-responsive smRNAs in spike tissues of the TGMS line. We also determined the expression profiles of target genes that encode transcription factors in response to cold stress. Interestingly, the expression of cold stress-responsive smRNAs integrated in the auxin-signaling pathway and their target genes was largely noncorrelated. We investigated the tissue-specific expression of smRNAs using a tissue microarray approach. Our data indicated that miR167 and tasiRNA-ARF play roles in regulating the auxin-signaling pathway and possibly in the developmental response to cold stress. These data provide evidence that smRNA regulatory pathways are linked with male sterility in the TGMS line during cold stress.

Characterizing thermosensitive genic male sterile lines of rice

The cytoplasmic male sterility (CMS) system is widely used to develop rice hybrids. Although effective, this system is quite cumbersome to practice. Certain genic male sterile lines that revert to fertility under specific temperature are called thermosensitive genic male sterile (TGMS) lines. Deployment of the TGMS system to develop two-line hybrids has several advantages over the conventional CMS system, as it requires neither maintainers for seed multiplication nor restorers for producing hybrids. In addition, two-line hybrids may not suffer from possible negative effects of the sterility-inducing cytoplasm.

Exploiting heterosis using TGMS lines in intra- and intersubspecific hybridization

The performance of various thermo- sensitive genic male sterile (TGMS) lines was assessed in order to use them in two-line intra- and intersubspecific hybridization with wide compatibility lines. Some 26 lines comprising TGMS lines, indica accessions, and japonica with wide compatibility (WC) gene(s) were used to make 65 hybrids: 31 were indica/indica and 34 were indica/japonica (WC). Standard heterosis was recorded up to 58%. Seed yield (g plant -1 ) ranged from 2.3 to 31.3. The number of heterotic combinations was higher in intersubspecific cross combinations. Spikelet fertility ranged from 2.7 to 88.4% in intersubspecific crosses and from 3.6 to 96.0% in intrasubspecific crosses. The WC lines were not univer- sally compatible in all combinations.

Organization of actin cytoskeleton during meiosis I in a wheat thermo-sensitive genic male sterile line

BS366 is a thermo-sensitive male sterile line of wheat (Triticum aestivum L.) for two-line hybrid breeding, which exhibits aberrant meiotic cytokinesis under low temperature. Through transcriptome analysis, a possible regulatory role for plant actin cytoskeleton was suggested. However, the organization of actin cytoskeleton in meiosis has been poorly understood so far. Here, fixed microsporocytes during meiosis were labeled with tetramethylrhodamine isothiocyanate-phalloidin and 4',6-diamidino-2-phenylindole. Quantities of fluorescent micrographs were captured using a confocal microscope, including the transient state from metaphase to telophase. We observed that actin filaments were abundant in typical kariokinetic spindle, central spindle (parallel microtubules or actin fibers between two separated chromosomes in anaphase), and phragmoplast. Interestingly, we identified the Chinese lantern-shaped actin phragmoplast in wheat meiosis for the first time. Under low temperature, phragmoplast actin filaments were chaotic and normal cell plate failed to form. These data provide new insights into the organization of actin filaments during male meiosis of plant and support a role of actin cytoskeleton in bringing about thermo-sensitive male sterility in wheat.

Development of specific simple sequence repeat (SSR) markers for non-pollen type thermo-sensitive genic male sterile gene in rice (Oryza sativa L.)

A key factor of two-line hybrid rice production is the development of thermo-sensitive genic male sterility (TGMS) lines. In this study, a TGMS line showing non-pollen type thermo-sensitive genic male sterility was used. Crossing between TGMS line (female parent) and normal pollen varieties; CNT1 and PTT1 (male parent) were performed and F 1 and F 2 populations were develop for each crosses. The phenotypic segregation ratio, 3:1 (fertile: sterile pollens) observed in F 2 populations of TGMS/CNT1 and TGMS/PTT1 crosses confirmed that the non-pollen type thermo-sensitive genic male sterility is controlled by a single recessive gene. The bulk segregant analysis (BSA) using simple sequence repeat (SSR) markers were deployed to identify the location and genetic effect of this gene. We have generated new set of SSR markers to identify progenies carrying TGMS gene in a cross TGMS/PTT1. The TGMS gene was located on chromosome 2 with 0.0 cM distance from T2 marker. This TGMS gene was located in the same region of previous identified tmsX gene. It might be the same allele of this gene of 2 different lines. This marker could be used for marker assisted backcrossing to transfer TGMS gene for developing female parent in two line hybrid rice system. Key words: Hybrid rice, thermo-sensitive genic male-sterile, non-pollen type, molecular marker, marker assisted backcrossing.

Determination of genetic relationships among elite thermosensitive genic male sterile lines (TGMS) of rice (Oryza sativa L.) employing morphological and simple sequence repeat (SSR) markers

A set of morphological traits and SSR markers were used to determine the genetic relationship among 12 elite thermosensitive genic male sterile (TGMS) lines developed at three different research institutions of India. Agro-morphological data recorded on 20 morphological traits revealed a wide base of genetic variation and a set of four morphological traits could distinguish most of the TGMS lines. Analysis with 30 SSR markers (20 EST-SSRs and 10 genomic SSRs) revealed 27 markers to be polymorphic, amplifying a total of 83 alleles. Each SSR marker amplified 2-6 alleles with an average of 2.76 alleles per marker and a PIC value varying from 0.54 to 0.96. Cluster analysis based on SSR and morphological data clearly differentiated the lines according to their source of origin. Correlation analysis between morphological and molecular data revealed a very poor association (r = 0.06), which could be attributed to selection pressure, genetic drift, sampling error and unknown relationship among related lines. The SSR markers discriminated the genotypes distinctly and quantified the genetic diversity precisely among the TGMS lines. Data on the yield per plant indicated that the genotypes grouping under a similar cluster showed same heterotic behaviour as compared to the genotypes from different clusters when crossed to similar pollinators.

Prediction of hybrid performance based on the genetic distance of parental lines in two-line rice (Oryza sativa L.) hybrids

A total of six TGMS (thermosensitive genic male sterile lines) and nine pollinator lines were subjected to molecular characterization using 48 genome-wide SSR (simple sequence repeat) markers. Cluster analysis revealed clear differentiation among the TGMS lines according to their source of origin. The SSR-based genetic distance between the hybrids of the parental lines ranged from 0.36 to 0.79 suggesting a high degree of genetic divergence. Among a set of 54 hybrids generated using parental lines, 32 showed better parent heterosis (+21.7%) while 19 showed mid-parent heterosis (+15.0%). For the trait yield per plant genetic distance (GD) was negatively correlated with F1 performance (r = −0.202), mid-parent heterosis (r = −0.325*; P < 0.05), and better parent heterosis (r = −0.261), while it was positively correlated with specific combining ability (r = 0.042). Based on the grouped genetic distance (GGD), the hybrid combinations were divided into four groups. The GGD showed linear correlation with hybrid performance within the group (GGD = 40–50: r = −0.07; GGD = 70–80: r = 0.32). This information can be utilized in the development of higher yielding, two-line rice hybrids through selection of intermediately diverse parental lines using GGD.

Cold stress contributes to aberrant cytokinesis during male meiosis I in a wheat thermosensitive genic male sterile line

The male sterility of a wheat thermosensitive genic male sterile (TGMS) line is strictly controlled by temperature. When the TGMS line BS366 was exposed to 10 °C from the pollen mother cell stage to the meiosis stage, a few pollen grains were formed and devoid of starch. We report here a large-scale transcriptomic study using the Affymetrix wheat GeneChip to follow gene expression in BS366 line anthers in response to cold stress. Notably, many cytoskeletal signaling components were gradually induced in response to cold stress in BS366 line anthers. However, the cytoskeleton-associated genes that play key roles in the dynamic organization of the cytoskeleton were dramatically repressed. Histological studies revealed that the separation of dyads occurred abnormally during male meiosis I, indicating defective male meiotic cytokinesis. Fluorescence labelling and subcellular histological observations revealed that the phragmoplast was defectively formed and the cell plate was abnormally assembled during meiosis I under cold stress. Based on the transcriptomic analysis and observations of characterized histological changes, our results suggest that cold stress repressed transcription of cytoskeleton dynamic factors and subsequently caused the defective cytokinesis during meiosis I. The results may explain the male sterility caused by low temperature in wheat TGMS lines.

Heterosis for Photosynthetic and Morphological Characters in F1 Hybrid Rice from a Thermo-Sensitive Genic Male Sterile Line at Different Growth Stages

Heterosis for Photosynthetic and Morphological Characters in F1 Hybrid Rice from a Thermo-Sensitive Genic Male Sterile Line at Different Growth Stages

Genetic analysis and mapping of a thermosensitive genic male sterility gene,tms6(t), in rice (Oryza sativaL.)

Thermosensitive genic male-sterile (TGMS) rice is widely used in hybrid rice production. Because of its specific temperature requirement, it can be used only in a narrow rice-growing zone in Asia. A newly discovered TGMS line, G20S, has an opposite phenotype compared with normal TGMS lines. G20S is completely sterile when the temperature is lower than 29.5 degrees C. Thus, it can be widely used in a larger area. Genetic analysis indicated that the sterility of G20S was controlled by a single recessive gene that was named tms6(t). An F2 population from the cross between G20S and Jing226 was developed and used for molecular mapping of the tms6(t) gene. The simple sequence repeat technique, combined with bulked segregant analysis, was used to screen markers linked to the target gene, and linkage markers such as RM216, RM2504, RM3152, and RM4455 were identified. These markers are all located on chromosome 10; therefore, tms6(t) was subsequently mapped on chromosome 10. Co-segregation analysis using the F2 population showed that two of these markers, RM3152 and RM4455, were closely linked to tms6(t) at distances of 3.00 cM and 1.10 cM, respectively. Additionally, integration of the genetic and physical maps showed that there were 15 BAC/PAC clones overlapping between RM3152 and RM4455, and tms6(t) was mapped to an interval of 1455 kb.

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 degrees 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.

Isolation and Characterization of a Microsporocyte-Specific Gene, OsMSP, in Rice

A spikelet-specific cDNA clone, OsMSP (Oryza sativa meiosis serine protease), was isolated from a rice cDNA library constructed by suppression subtraction hybridization (SSH). Its expression level was significantly reduced in the thermo-sensitive genic male-sterile rice line at sterile temperature (30/26°C, day/night) compared with that at fertile temperature (23/21°C, day/night) conditions. In contrast, no significant differences were observed in the normal fertile line under the above two sets of temperature conditions. The cDNA is 2,867 bp in length with an open reading frame (ORF) encoding 837 amino acid residues. Northern blots revealed that OsMSP transcripts were specifically present in spikelets at meiosis, and reached the highest level at early microspore stage. mRNA in situ hybridization confirmed that OsMSP was a rice microsporocyte-specific gene and its expression was limited to early microspore developmental stage. BLAST and phylogenetic analyses revealed that OsMSP shared high similarities with subtilisin-like serine protease genes from other organisms at both nucleotide and amino acid levels.

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 F(2) 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.

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°C, 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°C, 24°C and 26°C, and the length of elongated uppermost internode increased with the decreasing temperatures. At 28°C, 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.