Photoperiod-sensitive Genic Male Sterility Research Articles

The regulatory role of CARBON STARVED ANTHER-mediated photoperiod-dependent male fertility in rice.

Environmental signals, especially daylength, play important roles in determining fertility in photoperiod-sensitive genic male sterile (PGMS) lines that are critical to sustain production of high-yielding hybrid rice (Oryza sativa) varieties. However, the mechanisms by which PGMS lines perceive changes in photoperiod and transmit those signals to elicit downstream effects are not well understood. In this study, we compared the transcriptomes from the leaves and anthers of carbon starved anther (csa), a PGMS line, to wild-type (WT) tissues under different photoperiods. Components of circadian clock in the leaves, including Circadian Clock-Associated 1 and Pseudo-Response Regulator (PRR95), played vital roles in sensing the photoperiod signals. Photoperiod signals were weakly transduced to anthers, where gene expression was mainly controlled by the CSA allele. CSA played a critical role in regulating sugar metabolism and cell wall synthesis in anthers under short-day conditions, and transcription of key genes inducing csa-directed sterility was upregulated under long-day (LD) conditions though not to WT levels, revealing a mechanism to explain the partial restoration of fertility in rice under LD conditions. Eight direct targets of CSA regulation were identified, all of which were genes involved in sugar metabolism and transport (cell wall invertases, SWEETs, and monosaccharide transporters) expressed only in reproductive tissues. Several hub genes coordinating the effects of CSA regulation were identified as critical elements determining WT male fertility and further analysis of these and related genes will reveal insights into how CSA coordinates sugar metabolism, cell wall biosynthesis, and photoperiod sensing in rice anther development.

HMS1 interacts with HMS1I to regulate very-long-chain fatty acid biosynthesis and the humidity-sensitive genic male sterility in rice (Oryza sativa).

Environment-sensitive genicmale sterility (EGMS) lines are used widely in two-line hybrid breeding in rice (Oryza sativa). At present, photoperiod-sensitive genic male sterility (PGMS) lines and thermo-sensitive genic male sterility (TGMS) lines are predominantly used in two-line hybrid rice, with humidity-sensitive genic male sterility (HGMS) lines rarely being reported. Here, it is shown that HUMIDITY-SENSITIVE GENIC MALE STERILITY 1 (HMS1), encoding a β-ketoacyl-CoA synthase, plays key roles in the biosynthesis of very-long-chain fatty acids (VLCFAs) and HGMS in rice. The hms1 mutant displayed decreased seed setting under low humidity, but normal seed setting under high humidity. HMS1 catalyzed the biosynthesis of the C26 and C28 VLCFAs, contributing to the formation of bacula and tryphine in the pollen wall, which protect the pollen from dehydration. Under low-humidity conditions, hms1 pollen showed poor adhesion and reduced germination on the stigmas, which could be rescued by increasing humidity. HMS1-INTERACTING PROTEIN (HMS1I) interacted with HMS1 to coregulate HGMS. Furthermore, both japonica and indica rice varieties with defective HMS1 exhibited HGMS, suggesting that hms1 potentially could be used in hybrid breeding. The results herein reveal the novel mechanism of VLCFA-mediated pollen wall formation, which protects pollen from low-humidity stress in rice, and has a potential use in hybrid crop breeding.

Generating Photoperiod-Sensitive Genic Male Sterile Rice Lines with CRISPR/Cas9.

Obtaining photoperiod-sensitive genic male sterility (PGMS) lines is one of the most important steps in two-line hybrid rice breeding. Traditionally, such lines were screened and developed with a classic rice breeding system under both long-day and short-day conditions. The isolation and backcross process used for this could easily last for more than 3 years with a very low success rate. Here, we describe a straightforward method for generating csa-based PGMS lines by using the CRISPR-Cas9 technology in rice.

Genetic and molecular characterization of photoperiod and thermo-sensitive male sterility in rice.

A review on photoperiod and temperature-sensitive genic male sterility in rice. Male sterility in plants, facilitating the development of hybrid crops, has made great contribution to crop productivity worldwide. Environment-sensitive genic male sterility (EGMS), including photoperiod-sensitive genic male sterility (PGMS) and temperature-sensitive genic male sterility (TGMS), has provided a special class of germplasms for the breeding of "two-line" hybrids in several crops. In rice, the finding of the PGMS NK58S mutant in 1973 started the journey of research and breeding of two-line hybrids. Genetic and molecular characterization of these germplasms demonstrated diverse genes and molecular mechanisms of male sterility regulation. Two loci identified from NK58S, PMS1 and PMS3, both encode long noncoding RNAs. A major TGMS locus, TMS5, found in the TGMS line Annong S-1, encodes an RNase Z. A reverse PGMS mutant carbon starved anther encodes an R2R3 MYB transcription factor. Breeding efforts in the last three decades have resulted in hundreds of EGMS lines and two-line hybrids released to rice production, which have greatly elevated the yield potential and grain quality of rice varieties. The enhanced molecular understanding will offer new strategies for the development of EGMS lines thus further improving two-line hybrid breeding of rice as well as other crops.

Optimization of the Cry2Aa gene and development of insect-resistant and herbicide-tolerant photoperiod-sensitive genic male sterile rice

In this study, an optimized Cry2Aa gene was obtained after codon optimization based on the preferred codons in rice. The novel fusion gene Cry2Aa<sup>#</sup> was designed by adding the sequence coding the signal peptide of PR1a at the 5' end and the endoplasmic reticulum retention signal peptide KDEL at the 3' end of the optimized Cry2Aa gene, respectively. The Cry2Aa<sup>#</sup> and Bar genes were transformed into 4008S, a photoperiod-sensitive genic male sterile (PGMS) line in rice, using the Agrobacterium transfer method. A total of 65 regenerated plantlets confirmed by PCR were produced, in which eight transgenic lines had single-copy insertions as confirmed by Southern blot analysis. The high variability of Cry2Aa<sup># </sup>gene expression was observed among independent transgenic lines with single-copy insertion, and the spatiotemporal difference of Cry2Aa protein expression was discovered in each transgenic line. The results showed that the transgenic lines were highly resistant to glufosinate, rice leaf roller and striped stem borer, which not only confirmed the effective optimization of the Cry2Aa gene but also produced a useful germplasm for breeding insect-resistant and herbicide-tolerant hybrid rice varieties.

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.

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.

English

In photoperiod-sensitive genic male sterile rice (Oryza sativa L.) pollen abort in long day and high temperature, and revert to fertility in short day length and low temperature growth conditions. This type of control of rice plant fertility can facilitate production of hybrid rice using two lines (photoperiod-sensitive genic male sterile (PGMS) and restorer) system. Our objective in this study was to determine anatomical changes in PGMS rice pollen cells induced by long day length and high temperature growth conditions that are responsible for the cell abortion. All materials in this study were sown at Zhejiang University research field at Hangzhou, China 30°15’ N. Seeds of ZAU11S106, a PGMS rice line, were sown so as to obtain two bulks, 1) Extreme sterile pollen bulk and 2) fertile pollen bulk. Rice plants for extreme sterile pollen bulk were sown on March 18th and those for fertile pollen bulk on July 15th, so that they flowered under 13 h or more and under less than 13 h respectively of day light. Transmission electronic microscopes were used to study histological sections of anthers while light microscopes were used to examine whole pollen cells. The tapetal layers and the cytoplasm of the sectioned ZAU11S106 rice anthers from extreme sterile pollen bulk were deformed but those from fertile pollen bulk were normal when compared to the control rice lines ZAU11F121 and Xusui163. Pollen cells from anthers of extreme sterile pollen bulk had eroded exine and intine, missing or damaged nucleus, and disintegrating cytoplasm that lead to cell abortion. Our conclusion is that, cell abortion observed in extreme sterile pollen bulk of photoperiod genic male sterile rice, display “programmed cell death–like behavior”.   Key words: Pollen cell abortion, photoperiod genic male sterile rice, programmed cell death-like, tapetum.

Premature Tapetum Degeneration: a Major Cause of Abortive Pollen Development in Photoperiod Sensitive Genic Male Sterility in Rice

Photoperiod-sensitive genic male-sterile (PSGMS) rice (Oryza sativa L.), a natural mutant found in the rice cultivar Nongken 58, is very useful for the development of hybrid rice cultivars. Despite its widespread use in breeding programs, the initial stage of the abortive development of PSGMS rice and the possible cytological mechanisms of pollen abortion have not been determined. In the present study, a systematic cytological comparison of the anther development of PSGMS rice with its normal fertile counterpart is conducted. The results show that pollen abortion in PSGMS rice first occurs before the pollen mother cell (PMC) stage, and continues during the entire process of pollen development until pollen degradation. The abortive process was closely associated with the abnormal behavior of the tapetum. Although tapetum degeneration in PSGMS rice initiates already at the PMC stage, it proceeds slowly and does not complete until the breakdown of the pollen. Such cytological observations were supported by the results of the TUNEL (TdT-mediated dUTP Nick End Labeling) assay, which detects DNA fragmentation resulting from programmed cell death (PCD), indicating that the premature tapetum degeneration is in the process of PCD.

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.

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 F 2 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.

Cytological study and PCD assay on pollen development of photoperiod sensitive genic male sterile rice

A systematic cytological comparison of the anther development of photoperiod sensitive genic male sterile (PSGMS) rice with its normal fertility counterpart was conducted. The results showed that pollen abortion in PSGMS rice occurred first no later than the pollen mother cell (PMC) stage and continued during the entire process of pollen development till pollen degradation. This abortive process was closely associated with the abnormal behavior of tapetum. Although tapetum degeneration in the PSGMS rice initiated as early as at the PMC stage, it proceeded slowly and did not complete until the breakdown of the pollen, in sharp contrast to the rapid disintegration of the tapetal layer during the late microspore to the bicellular pollen stage in the fertile rice. Such cytological observation was supported by the results of the TUNEL (TdT2 mediated dU TP Nick End Labeling) assay that detectsDNA fragmentation resulting from programmed cell death (PCD), indicating that the tapetum degeneration occurs in the process of PCD.

Daylength responses of various morphogenetic events after panicle initiation in late japonica rice (Oryza sativa L. ssp. japonica)

Plant morphogenesis is an intimate cooperation between environmental signals and a genetically-encoded developmental programme. Although all stages subsequent to floral induction may be influenced by daylength, the information available has come from diverse species during different morphogenetic events. This makes it difficult to investigate how photoperiodic signalling is integrated into various steps of the developmental programme. Here, we report that, in late japonica rice lines, morphogenetic events, including 12th leaf, axis of the main panicle, awn of the glum, anthers, elongation rate of internodes, and pollen fertility of the photoperiod-sensitive genic male-sterile (PGMS) rice, are significantly affected by daylength after panicle initiation. These data indicate that daylength affects many morphologic events, and that changes in morphology are mainly determined by the characteristics of the events themselves. These findings lay a foundation for future investigations into how potentially common photoperiodic signalling system(s) are integrated with diversified developmental events. In addition, we discussed the essential nature of PGMS rice.

Strategy and utilization of a herbicide resistance gene in two-line hybrid rice

Hybrid rice plays an important role in China's aim to improve rice production as it accounts for some 50% of rice planting area but produces about 60% of the total rice grain. However, the existing three-line system used in hybrid rice production has its limitations. The two-line system, which makes use of photoperiod-sensitive genic male-sterile (PGMS) and thermo-sensitive genic male-sterile (TGMS) lines to generate the male-sterile parental line, was developed to overcome some of these limitations. The sterility of the male-sterile line of two-line hybrid rice, however, fluctuates when the temperature-sensitive phase of fertility encounters abnormal low temperatures during hybrid seed production, which induces selfing and decreases the purity of hybrid. We describe here the strategy of utilizing a herbicide resistance gene in two-line hybrid rice to eliminate this fluctuation in the sterility of the P/TGMS lines during hybrid seed production and reports the development of the herbicide resistance restorer line Bar68-1 and its herbicide-resistant early season hybrid rice Xiang125s/Bar68-1. When the restorer line and its derived hybrid are herbicide resistant, the selfed seeds can be removed easily from the hybrid by herbicide spraying. A herbicide resistance gene bar was transferred into a restorer line by particle bombardment. The resulting transgenic restorer line Bar68-1 and its hybrid Xiang125 s/Bar68-1 inherited stable herbicide resistance. The purity of Xiang125s/Bar68-1 was increased by spraying the seed bed with herbicide, which resulted in a significant increase in yield, grain quality, and disease resistance in comparison to the controls in a regional trial.

Use of herbicide-resistant genic male sterility in hybrid rice seed production

One of the difficult problems in hybrid rice seed production is the low outcrossing frequency and requirement for much labor to produce hybrid seeds. In order to simplify the process of hybrid rice seed production, herbicide-resistant photoperiod sensitive genic male sterile (HRPGMS) rice was utilized in this study. The herbicide resistance gene bar was transferred into the photoperiod sensitive genic male sterile (PGMS) rice 920S by Agrobacterium-mediated transformation and the HRPGMS line YA3530ms with good agronomic characteristics was bred by applying conventional pedigree breeding technique. The seeds of HRPGMS and pollen parent were mixed with the ratio of 4:1 in weight, and were sowed in seedling box. The mixed seedlings of HRPGMS and pollen parent grown for 30 days were transplanted by the small transplanting machine in the field. The herbicide glufosinate ammonium was sprayed at 7 days after flowering to kill all the plants of pollen parent, whereas hybrid seeds were harvested from the survived HRPGMS parent at maturity. The outcrossing frequency of HRPGMS line from two combinations in 2002 and from five combinations in 2004 were compared with a control cultivated by the conventional 2-line system. As the result, the mean outcrossing frequency in HRPGMS of the treatments were 10.6–24.5% compared with 5.5% in PGMS of the control in 2002, and that were 24.7–32.0% compared with 7.5% in the control in 2004. Consequently, using HRPGMS in two-line system was proved to be a new method that would simplify the process of hybrid rice seed production and to increase outcrossing frequency without any artificial supplementary pollination processes.

Rapid genome evolution in Pms1 region of rice revealed by comparative sequence analysis

Pms1, a locus for photoperiod sensitive genic male sterility in rice, was identified and mapped to chromosome 7 in previous studies. Here we report an effort to identify the candidate genes for Pms1 by comparative sequencing of BAC clones from two cultivars Minghui 63 and Nongken 58, the parents for the initial mapping population. Annotation and comparison of the sequences of the two clones resulted in a total of five potential candidates which should be functionally tested. We also conducted comparative analysis of sequences of these two cultivars with two other cultivars, Nipponbare and 93-11, for which sequence data were available in public databases. The analysis revealed large differences in sequence composition among the four genotypes in the Pms1 region primarily due to retroelement activity leading to rapid recent growth and divergence of the genomes. High levels of polymorphism in the forms of indels and SNPs were found both in intra-and inter-subspecific comparisons. Dating analysis using LTRs of the retroelements in this region showed that the substitution rate of LTRs was much higher than reported in the literature. The results provided strong evidence for rapid genomic evolution of this region as a consequence of natural and artificial selection.

Localization of pms3, a gene for photoperiod-sensitive genic male sterility, to a 28.4-kb DNA fragment

Photoperiod-sensitive genic male-sterile (PSGMS) rice, in which pollen fertility is regulated by day-length, originally arose as a natural mutant in the rice cultivar Nongken 58 (Oryza sativa ssp. japonica). Previous studies identified pms3 on chromosome 12 as the locus of the original PSGMS mutation. In this study we have assigned the pms3 locus to a 28.4-kb DNA fragment by genetic and physical mapping. A cross between Nongken 58S (PSGMS line) and DH80 was used to produce an F2 population of about 7000 plants, from which 892 highly sterile individuals were obtained for recombination analysis. By analyzing recombination events in the sterile individuals using a total of 157 RFLP probes from a BAC contig covering the pms3 region, the pms3 locus was localized to a sub-region of less than 1.7 cM. Further analysis of recombination events using 49 additional probes isolated from this sub-region identified markers flanking the pms3 region on each side; these markers are only 28.4-kb apart. Sequence analysis of this fragment predicted the presence of five ORFs, found high homology with two ESTs in public databases, and detected three SNPs between the mutant and the wild-type parents, which may be helpful for identifying a candidate gene for pms3.

Development of TGMS lines and two-line rice hybrids through a shuttle breeding program between IRRI and China

Based on the discovery of photoperiod-sensitive genic male sterile (PGMS) rice mutant Nongken 58S (Shi 1981), rice scientists in China began work on the two-line system of hybrid rice breeding in the early 1980s. Subsequently, some thermosensitive genic male sterile (TGMS) rice mutants, such as Annong S-1 and 5460S, were developed in 1988 through screening of breeding materials and radiation, respectively (Lu et al 1998). The two-line system was established in China by using PGMS and TGMS lines. This method was considered more efficient than the thre-line system in exploiting heterosis to increase yield.

Isolation ofosRACD gene encoding a small GTP-binding protein from rice

Using an improved version of mRNA differential display technology, we have obtained a differentially displayed fragment RDP-8. Homologous comparison indicated that the fragment RDP-8 has high homology with the gene encoding maize small GTP-binding protein. By screening cDNA library of the rice Nongken 58N panicle using the newly obtained fragment RDP-8 as probe, we further found the full-length cDNA ofosRACD gene that encodes a rice small GTP-binding protein. As compared with maizeRACD gene, the osRACD of rice shows remarkable homology in both nucleotide sequence and amino acid sequence, 88% and 97% respectively. Evidence from RT-PCR study indicates thatosRACD gene is related to photoperiod fertility conversion of photoperiod sensitive genic male sterility (PSGMS) rice.

Identification of an 85-kb DNA fragment containing pms1, a locus for photoperiod-sensitive genic male sterility in rice.

Photoperiod-sensitive genic male-sterile rice has a number of desirable characteristics for hybrid rice production. Previous studies identified pms1, located on chromosome 7, as a major locus for photoperiod-sensitive genic male sterility. The objective of this study was to localize the pms1 locus to a specific DNA fragment by genetic and physical mapping. Using 240 highly sterile individuals and a random sample of 599 individuals from an F2 population of over 5000 individuals from a cross between Minghui 63 and 32001S, we localized the pms1 locus by molecular marker analysis to a genetic interval of about 4 cM, 0.25 cM from RG477 on one side and 3.8 cM from R1807 on the other side. A contig map composed of seven BAC clones spanning approximate 500 kb in length was constructed for the pms1 region by screening a BAC library of Minghui 63 DNA using RFLP markers and chromosomal walking. Analysis of recombination events in the pms1 region among the highly sterile individuals reduced the length of the contig map to three BAC clones. Sequencing of one BAC clone, 2109, identified two SSR markers located 85 kb apart in the clone that flanked the pms1 locus on both sides, as indicated by the distribution of recombination events. We thus concluded that the pms1 locus was located on the fragment bounded by the two SSR markers.