Thermosensitive Genetic Male Sterility Research Articles

Transcriptomics Analysis on Fertility Conversion in Thermosensitive Genetic Male Sterility Line Zhu1S under High Temperature

Zhu1S is a thermosensitive genic male-sterile (TGMS) line of rice possessing outstanding combining ability and low critical temperature, which has been extensively utilized as a female parent in two-line hybrid ricebreeding. However, the fertility of Zhu 1S during hybrid seed production is frequently affected by high temperature, thus leading to its fertility alteration and aborted hybrid seed production. To understand its fertility conversion mechanism under high temperature, we employed transcriptomics analyses on the anthers of young panicles of Zhu 1S during the fertility alternation sensitivity stage under high (Zhu 1S-H) and low (Zhu 1S-L) temperatures. The results showed that a total of 1119 differentially expressed genes (DEGs) were identified between Zhu 1S-H and Zhu1S-L anthers, including 680 up-regulated and 439 down-regulated genes. Bioinformatics analysis of these DEGs revealed that the high temperature induction caused fertility-sterility conversion in Zhu1S, mainly by decreasing the mRNA abundances of important genes closely related to plant hormone and MAPK signal pathway and transcriptional regulation factors, thereby impeding the growth and development of the anther of Zhu 1S, which ultimately affected the fertility transition of Zhu 1S under high temperature. The protein–protein interaction network analysis indicates that transcription factor OsTIFY11C possibility plays a central role in the fertility transition of Zhu 1S under high temperature.The present studies offer a theoretical foundation for further research into the molecular mechanism underlying fertility conversion in TGMS line Zhu 1S.

Characterization of fertility alteration and marker validation for male sterility genes in novel PTGMS lines hybrid rice.

Photoperiod and thermosensitive genetic male sterile (PTGMS) lines have become one of the main sources of global rice production increasing. This study was conducted to evaluate the fertility alteration and validate the male sterility genes using validation markers in novel Egyptian Indica and Japonica PTGMS lines under natural conditions. The study revealed that the new genetic male sterile lines belong to the type of photo–thermosensitive genetic male sterility (PTGMS). The fertility alteration of these lines has influenced by photoperiod and temperature interaction. The new PTGMS lines have three sensitive periods of fertility alteration; transformation, sterility, and fertility period. Furthermore, the sensitive stage of fertility transformation might be from secondary branch primordial to pollen mother cells (PMC) meiosis. Under the natural Sakha condition, the new PTGMS lines were stable sterile under the condition of day length upper 13,75 h and temperature over 25 °C, while its convert to fertile under day length under 13 h, and temperature lower than 24 °C. The co-dominant markers identified the pms3 and tms5 genes in the new PTGMS lines, indicated that the fertility alteration in these lines controlled by photoperiod and thermosensitive stages.

A quantitative proteomic analysis of the molecular mechanism underlying fertility conversion in thermo-sensitive genetic male sterility line AnnongS-1

BackgroundThermo-sensitive genetic male sterile (TGMS) lines have been widely used in two-line hybrid rice breeding. The two-line hybrids have increased rice yields substantially. However, the effect of environmental temperatures on the fertility conversion is still not fully clear. In this study, we performed a tandem mass tag (TMT)-based proteomic analysis on the anthers of the TGMS line AnnongS-1 grown under permissive (low) temperature (21 °C) and restrictive (high) temperature (> 26 °C) conditions in an attempt to explore the effect of temperature on the fertility of the male sterile line.ResultsAfter the AnnongS-1 plants were induced under either permissive or restrictive conditions, morphological observations and I2-KI staining confirmed that the pollen grains formed under high temperature conditions were abortive while those formed under low temperature developed normally. In comparison to the plants grown under permissive conditions, the restrictive high-temperature conditions led to the differential accumulation of 89 proteins in the anthers, of which 46 were increased in abundance and 43 were decreased in abundance. Most of the subcellular compartments of the anther cells had one or more proteins that had been differentially accumulated, with the cytoplasm and chloroplast having the greatest accumulations. More than 40% of the differentially abundant proteins (DAPs) were enzymes involved in photosynthesis, energy metabolism, biosynthesis and catabolism of cellular components, metabolic regulation, defense and stress, etc. The DAPs related to protein metabolism accounted for the largest proportion (21.35%), followed by those related to defense and stress (12.36%), metabolic regulation (10.11%) and carbohydrate metabolism (8.99%), indicating that such biological processes in anther cells were more susceptible to high temperature stress.ConclusionsThe restrictive temperature induction caused fertility-sterility conversion in the TGMS line AnnongS-1 mainly by adversely affecting the metabolism of protein, carbohydrate and energy, and decreasing the abundances of important proteins closely related to defense and stress, thereby impeding the growth and development of the pollen and weakening the overall defense and ability to endure stress of AnnongS-1. These data are helpful for deepening our understanding of the molecular mechanism underlying fertility conversion in TGMS lines.

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

For the two-line hybrid rice system, pollen 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) controlling 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 pollen 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.

Pyramiding of thermosensitive genetic male sterility (TGMS) genes and identification of a candidate tms5 gene in rice

Three thermosensitive genetic male sterility (TGMS) genes – tms2, tgms and tms5 – were pyramided using linked microsatellite markers. Three TGMS donors, Norin PL 12 (tms2), SA2 (tgms) and DQ200047-21 (tms5) were utilized in generating crosses from which two-gene and three-gene pyramids possessing the RM11 allele of Norin PL 12, RM257 allele of SA2 and RM174 allele of DQ200047-21 were selected. All selected progenies were male-sterile at sterility-inducing conditions. In addition, rice SF21 was identified as a candidate tms5 gene because of its complete linkage with RM174. The 4,200-bp region was amplified from the TGMS line M105S and the two ends were sequenced. In silico analysis of partial nucleotide sequences showed that the region is similar to the SF21 pollen-specific gene of Arabidopsis and Helianthus. The M105S tms5 sequence was also compared to the SF21 sequence from the International Rice Genome Sequencing Project (IRGSP) database.

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

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

Microsatellite Markers Flanking the tms2 Gene Facilitated Tropical TGMS Rice Line Development

The use of thermosensitive genetic male sterility (TGMS) in the development and production of rice (Oryza sativa L.) hybrids is an alternative to the cytoplasmic‐genetic male sterility (CMS) system. This study aimed to develop TGMS lines with aromatic Thai rice background by molecular marker‐aided breeding. Four microsatellite markers (RM2, RM10, RM11, and RM214) on chromosome 7 in the vicinity of the TGMS gene tms2 and showing polymorphism between the two parents were used in genotyping the mapping population consisting of 157 F2 plants derived from a cross between Norin PL12 (a TGMS line from Japan) and KDML 105 (a popular aromatic Thai rice cultivar). The RM11 marker was approximately 5 centimorgans (cM) from tms2 while RM2 was approximately 16 cM from it. In this F2 population, the accuracy of selecting sterile plants with RM2 and RM11 markers was 92 and 97%, respectively. In three backcrosses, the accuracy of selection with markers for either homozygous or heterozygous plants was higher than 90% with RM2. Using RM11, we obtained 89% accuracy for selecting homozygous fertile plants and 59% accuracy for selecting heterozygous plants. The results demonstrated that microsatellite markers were powerful in screening large breeding populations, and these markers facilitated selection for plants possessing the tms2 in an early stage of the crop and without exposing the materials to the required temperature for TGMS gene expression. Three TGMS lines with aromatic Thai rice background were developed and showed complete pollen sterility when maximum temperature was higher than 30°C, 1 to 2 wk after panicle initiation. Up to 77% spikelet fertility was observed when these lines were exposed at temperature below 30°C during the critical stage.

Agro-morphological characterization of ovary culture-derived plants of rice (Oryza sativa L.)

Plants derived from unpollinated ovary culture of ten rice genotypes showed significant variability in agro-morphological characteristics. The ovary-derived plant (H1) populations were completely haploid, doubled haploid or haploid-doubled haploid mixture. Haploids had very drastic reduction in plant height, panicle length, grain length, breadth and number and spikelet fertility (0.0%–2.1%). Doubled haploids from the hybrid of UPRI 95–121 × UPRI 95–165 were normal with fertility ranging between 69.6% and 97.7%. A genetic segregation in ratio of 1:1 was observed for five pigmentation characters in the H1 population derived from hybrid UPRI 95–122 × UPRI 95–165. Plant height showed the largest coefficient of variability (28.5%) followed by the number of spikelets per panicle (24.2%), number of grains per panicle (22.0%), percent seed set (9.2%) and panicle length (9.0%). The range of variation in the H1 population from fully fertile hybrid PMS 2A (CMS) × IR 31802 (restorer) was similar to its corresponding F2 population for plant height, spikelet fertility and number of grains/panicle. A single clone of plants from the cultivar BG 1321 exhibited complete male sterility but normal female fertility when pollinated with other varieties. Ovary-derived plants from the CMS lines PMS 2A and IR 58025A showed stable male sterility and those from thermosensitive genetic male sterile line UPRI 95–140 showed thermosensitive genetic male sterility. These lines have potential in the hybrid breeding program and are being currently exploited.

A TGMS-linked nuclear DNA marker as originated from the mitochondrial genome in rice (Oryza sativa L.)

A nuclear RAPD marker, OPF182600, was linked to a thermosensitive genetic male sterility (TGMS) gene (tms3) in a rice mutant line IR32364TGMS. Segregation analysis in two mapping populations and reciprocal cross analysis involving different genotypes indicated, however, that this marker was maternally inherited. The RAPD marker was then cloned as pF18 and partially sequenced from both ends of the insert. Sequence analysis showed that pF18 was homologous to mitochondrial genes, cob2 and nad1. Evidence was provided that the cob2 DNA segment near pF18 was genetically linked to tms3. Thus pF18 could be the result of integration of a piece of mitochondrial DNA segment into the nuclear genome. The transferred DNA segment included at the least cob2, a portion of nad1, and intergenic sequences. The presence of intron and intergenic sequences in the transferred DNA fragment indicated that DNA was the transfer intermediate. The relationship between the transferred DNA and tms3 is discussed.

A TGMS-linked nuclear DNA marker as originated from the mitochondrial genome in rice (Oryza sativa L.)

A nuclear RAPD marker, OPF182600, was linked to a thermosensitive genetic male sterility (TGMS) gene (tms3) in a rice mutant line IR32364TGMS. Segregation analysis in two mapping populations and reciprocal cross analysis involving different genotypes indicated, however, that this marker was maternally inherited. The RAPD marker was then cloned as pF18 and partially sequenced from both ends of the insert. Sequence analysis showed that pF18 was homologous to mitochondrial genes, cob2 and nad1. Evidence was provided that the cob2 DNA segment near pF18 was genetically linked to tms3. Thus pF18 could be the result of integration of a piece of mitochondrial DNA segment into the nuclear genome. The transferred DNA segment included at the least cob2, a portion of nad1, and intergenic sequences. The presence of intron and intergenic sequences in the transferred DNA fragment indicated that DNA was the transfer intermediate. The relationship between the transferred DNA and tms3 is discussed.

Development of a Thermosensitive Genetic Male Sterile Line for Hybrid Breeding through Ovary Culture in Rice (Oryza sativa L.)

Unpollinated young ovaries from the F1 plants of the cross between UPRI 95-140, a TGMS donor, and UPRI 95-117, a good plant type line of indica rice were pretreated at 8°C for 14 days and cultured onN6 medium supplemented with lactoprotein hydrolysate 500 mg l-1 2,4-D 4.0 mg l-1 NAA 2.0 mg l-1 and BA 1.0 mg l-1. Five calli with 0.5% of callus induction were obtained. Four of these calli gave complete green plantlets possible of spontaneous dihaploids while the remaining one gave partially green plantlets onMS plant regeneration medium supplemented with casein acid hydrolysate 500 mg l-1, NAA 0.5 mg l-1 and BA 1-5 mg l-1. One of the regenerated green plantlets was identified with thermosensitive genetic male sterility (TGMS) trait. The spikelet fertility transformation of this plant was closely related to mean minimum daily and mean average daily temperatures of the 15-d thermosensltive stage prior to 5 days of heading. It presented complete spikelet sterility between May 1 and Sept 5 and 0.5 to 12.3% and 0.5 to 41.5% of seed set between Sept 18 and Nov 1, 1995 and March 10 to april 27, 1996, respectively at Pantnagar in the northern India. And the line in H2 present homogenous performance with optimum spikelet fertility transformation in Wet season, 1996. This line is being used to develop two-line heterotic hybrids.

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

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

Classification of environmentally induced genetic male sterile lines of rice based on their fertility responses to photoperiod and temperature

SUMMARYThe use of environmentally induced genetic male sterile (EGMS) rice could alter the development of hybrid rice from a three-line system to a two-line system. It is critical for the utilization of EGMS rice to determine which are the main environmental factors influencing fertility changes. Fertility responses to photoperiod (P) and temperature (T) were studied in 101 EGMS rice lines under nine controlled regimes combining three photoperiods (15·0, 14·0 and 12·5 h)x three temperatures (30·1, 24·1 and 23·1 °C). According to the variance analysis of seed-setting data, 96% of the total EGMS lines studied could be divided into three types as follows: (1) photoperiod-sensitive genetic male sterility (PGMS) characterized statistically by significant (P < 0·05) P and P × T interaction effects but by a non-significant T effect on fertility, (2) thermosensitive genetic male sterility (TGMS) by a significant T effect, a non-significant P effect and by either a significant or a non-significant P × T interaction effect on fertility, and (3) photo-thermosensitive genetic male sterility (P-TGMS) by only a significant P × T interaction effect on fertility. Among the japonica EGMS lines studied, PGMS, TGMS and P-TGMS accounted for 32·3, 9·7 and 51·6%, respectively. However, among the indica EGMS lines, no PGMS lines were detected and most of them were TGMS or P-TGMS (61·4 and 35·7%, respectively). The results indicate that the selection of indica PGMS lines of rice might be very difficult. The availability of different types of EGMS rice in two-line system hybrid rice is evaluated and the selection of an ideal model of response to photoperiod and temperature for indica EGMS is discussed.