Map-based cloning of a novel rice cytochrome P450 gene CYP81A6 that confers resistance to two different classes of herbicides

Development of hybrid rice has greatly contributed to increased yields during the past three decades. Two bentazon-lethal mutants 8077S and Norin8m are being utilized in developing new hybrid rice systems. When the male sterile lines are developed in such a mutant background, the problem of F1 seed contamination by self-seeds from the sterile lines can be solved by spraying bentazon at seedling stage. We first determined the sensitivity of the mutant plants to bentazon. Both mutants showed symptoms to bentazon starting from 100 mg/l, which was about 60-fold, lower than the sensitivity threshold of their wild-type controls. In addition, both mutants were sensitive to sulfonylurea-type herbicides. The locus for the mutant phenotype is bel for 8077S and bsl for Norin8m. Tests showed that the two loci are allelic to each other. The two genes were cloned by map-based cloning. Interestingly, both mutant alleles had a single-base deletion, which was confirmed by PCR-RFLP. The two loci are renamed bel ^a (for bel) and bel ^b (for bsl). The wild-type Bel gene encodes a novel cytochrome P450 monooxgenase, named CYP81A6. Analysis of the mutant protein sequence also revealed the reason for bel ^a being slightly tolerant than bel ^b . Introduction of the wild-type Bel gene rescued the bentazon- and sulfonylurea-sensitive phenotype of bel ^a mutant. On the other hand, expression of antisense Bel in W6154S induced a mutant phenotype. Based on these results we conclude that the novel cytochrome P450 monooxygenase CYP81A6 encoded by Bel confers resistance to two different classes of herbicides. Gang Pan and Xianyin Zhang contributed equally to this work.     

Identification of a cytochrome P450 hydroxylase, CYP81A6, as the candidate for the bentazon and sulfonylurea herbicide resistance gene, Bel, in rice

Bentazon and sulfonylureas have been used for selective control of broadleaf weeds and sedges in rice fields for more than 20 years. A bentazon and sulfonylurea susceptible mutant, bel, was previously identified for the purpose of allowing these herbicides to be used for removing false hybrids from hybrid rice. While this mutation has been used successfully in rice breeding, the genetic nature of bel is not known. Using 1,776 susceptible plants from a population of 10,000 F₂ individuals, we constructed a fine map for the Bel locus and delimited it to a 36-kb DNA fragment between two restriction fragment length polymorphism markers, L5 and P17. Bioinformatic analysis indicated that there are five genes within this interval, an ethylene-responsive OsER33 gene and four tandem repeats of cytochrome P450 genes designated as CYP81A5, CYP81A6, CYP81A7, and CYP81A8. Comparative sequencing could not find any differences in the coding regions of the OsER33, CYP81A5, CYP81A7, and CYP81A8 genes between the mutant bel and its wild-type progenitor W6154S, but did identify a single base guanine deletion at position +1,332 bp downstream from the translation start codon of CYP81A6. This deletion introduces a premature stop codon and leads to the loss of the heme-binding motif, which is essential for cytochrome P450 function because it contains an absolutely conserved cysteine that serves as the fifth ligand to the heme iron. CYP81A6 presumably functions as a hydroxylase for the detoxification of bentazon and sulfonylurea herbicides in rice. A gene-specific cleaved amplified polymorphic sequence marker and tightly linked flanking markers were developed that will be very useful for selection of the bel allele when transferred to photoperiod-/thermo-sensitive genic male sterility and CMS lines in hybrid rice breeding programs.     

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-1X), 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 F₁ and F_2:3 populations derived from the cross between DNS-1X 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 rtms1-D. It was narrower than the previously reported RTGMS genes rtms1 and tms6(t). The fertility conversion detected in the natural environment showed that DNS-1X was sterile below 28–30 °C; 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.     

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°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.     

Genetic characterization and fine mapping of a novel thermo-sensitive genic male-sterile gene tms6 in rice (Oryza sativa L.)

The application of genetic male sterility in hybrid rice production has great potential to revolutionize hybrid seed production methodology. The two-line breeding system by using thermo-sensitive genic male sterility (TGMS) has been discovered and successfully developed as a breeding strategy in rice. One TGMS gene was investigated by a spontaneous rice mutant line, Sokcho-MS, originated from a Korean japonica variety. It was shown that Sokcho-MS is completely sterile at a temperature higher than 27°C and/or lower than 25°C during the development of spikelets, but fertile at the temperature ranging from 25 to 27°C regardless of the levels of day-length. Genetic analysis and molecular mapping based on SSR, STS and EST markers revealed that a single recessive gene locus involved the control of genic male sterility in Sokcho-MS. By using an F₂ mapping population derived from a cross between Sokcho-MS and a fertile indica variety Neda, the new TGMS gene, designated as tms6, was mapped primarily to the long arm of chromosome 5 of Oryza sativa at the interval between markers E60663 (2.0 cM) and RM440 (5.8 cM). Subsequently, tms6 was fine mapped to the interval between markers RM3351 (0.1 cM) and E60663 (1.9 cM). As tms6 appeared to be independent of other mapped TGMS genes in rice, the genetic basis of Sokcho-MS was further discussed.     

Fine mapping and candidate gene analysis of the novel thermo-sensitive genic male sterility tms9-1 gene in rice

Key message

Fine mapping of the novel thermo-sensitive genic male sterility locus tms9 - 1 in the traditional TGMS line HengnongS-1 revealed that the MALE STERILITY1 homolog OsMS1 is the candidate gene.

Abstract

Photoperiod-thermo-sensitive genic male sterility (P/TGMS) has been widely used in the two-line hybrid rice breeding system. HengnongS-1 is one of the oldest TGMS lines and is often used in indica two-line breeding programs in China. In this study, our genetic analysis showed that the TGMS gene in HengnongS-1 was controlled by a single recessive gene that was non-allelic with the other TGMS loci identified, including C815S, Zhu1S and Y58S. Using SSR markers and bulked segregant analysis, we located the TGMS locus on chromosome 9 and named the gene tms9-1. Fine mapping further narrowed the tms9-1 loci to a 162 kb interval between two dCAPS markers. Sequence analysis revealed that a T to C substitution results in an amino acid change in the tms9-1 candidate gene (Os09g27620) in HengnongS-1 as compared to Minghui63. Sequencing of other rice accessions, including six P/TGMS lines, seven indica varieties and nine japonica varieties, showed that this SNP was exclusive to HengnongS-1. With multiple sequence alignment and expression pattern analyses, the rice MALE STERILITY1 homolog OsMS1 gene was identified as the candidate gene for tms9-1. Therefore, our study identified a novel TGMS locus and will facilitate the functional identification of the tms9-1 gene. Moreover, the markers linked to the tms9-1 gene will provide useful tools for the development of new TGMS lines by marker-assisted selection in two-line hybrid rice breeding programs.     

Seedling herbivory by slugs in a willow hybrid system: developmental changes in damage, chemical defense, and plant performance

We evaluated feeding preference and damage by the slug, Arion subfuscus, on seedlings of two willow species, Salix sericea and S. eriocephala, and their F₁ interspecific hybrids. Trays of seedlings were placed in the field and excised leaves were presented to slugs in choice tests. Slugs preferred feeding on and caused the most damage to S. eriocephala seedlings. S. sericea seedlings were least preferred and least damaged. F₁ hybrid seedlings were intermediate in preference and damage. Slug preference of and damage to these seedlings decreased over time, suggesting developmental changes in resistance. Seedlings were sampled for phenolic glycoside and tannin chemistry weekly to coincide with the field and laboratory experiments. Concentrations of phenolic glycosides and tannins increased linearly with seedling age, coincident with changes in slug preference and damage, indicating a developmental change in defense. Slug deterrence was not detected at low concentrations of salicortin when painted on leaves or discs, but both salicortin and condensed tannins deterred slug feeding at concentrations between 50 and 100 mg/g, levels found in adult willows. Seedling performance was related to damage inflicted by slugs. Due to lower levels of damage when exposed to slugs in the field, S. sericea plants had significantly greater biomass than S. eriocephala plants. Biomass of F₁ hybrids was equal to S. sericea when damaged. However, undamaged S. eriocephala and F₁ hybrid plants had the greatest biomass. Because F₁ hybrid seedlings performed as well as the most fit parent in all cases, slugs could be an important selective factor favoring introgression of defensive traits between these willow species.     

Photoperiod- and thermo-sensitive genic male sterility in rice are caused by a point mutation in a novel noncoding RNA that produces a small RNA

Photoperiod- and thermo-sensitive genic male sterility (PGMS and TGMS) are the core components for hybrid breeding in crops. Hybrid rice based on the two-line system using PGMS and TGMS lines has been successfully developed and applied widely in agriculture. However, the molecular mechanism underlying the control of PGMS and TGMS remains obscure. In this study, we mapped and cloned a major locus, p/tms12-1 (photo- or thermo-sensitive genic male sterility locus on chromosome 12), which confers PGMS in the japonica rice line Nongken 58S (NK58S) and TGMS in the indica rice line Peiai 64S (PA64S, derived from NK58S). A 2.4-kb DNA fragment containing the wild-type allele P/TMS12-1 was able to restore the pollen fertility of NK58S and PA64S plants in genetic complementation. P/TMS12-1 encodes a unique noncoding RNA, which produces a 21-nucleotide small RNA that we named osa-smR5864w. A substitution of C-to-G in p/tms12-1, the only polymorphism relative to P/TMS12-1, is present in the mutant small RNA, namely osa-smR5864m. Furthermore, overexpression of a 375-bp sequence of P/TMS12-1 in transgenic NK58S and PA64S plants also produced osa-smR5864w and restored pollen fertility. The small RNA was expressed preferentially in young panicles, but its expression was not markedly affected by different day lengths or temperatures. Our results reveal that the point mutation in p/tms12-1, which probably leads to a loss-of-function for osa-smR5864m, constitutes a common cause for PGMS and TGMS in the japonica and indica lines, respectively. Our findings thus suggest that this noncoding small RNA gene is an important regulator of male development controlled by cross-talk between the genetic networks and environmental conditions.     

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 F₂ 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. H. F. Peng, Z. F. Zhang and B. Wu contributed equally to this work.     

Molecular mapping of a rice gene conditioning thermosensitive genic male sterility using AFLP, RFLP and SSR techniques

The discovery and application of the thermosensitive genic male sterility (TGMS) system has great potential for revolutionizing hybrid seed production technology in rice. Use of the TGMS system in two-line breeding is simple, inexpensive, efficient, and eliminates the limitations associated with the cytoplasmic-genetic male sterility (CMS) system. An F₂ population developed from a cross between a TGMS indica mutant, TGMS–VN1, and a fertile indica line, CH1, was used to identify molecular markers linked to the TGMS gene and to subsequently determine its chromosomal location on the linkage map of rice. Bulk segregant analysis was performed using the AFLP technique. From the survey of 200 AFLP primer combinations, four AFLP markers (E2/M5–600, E3/M16–400, E5/M12–600, and E5/M12–200) linked to the TGMS gene were identified. All the markers were linked to the gene in the coupling phase. All except E2/M5–200 were found to be low-copy sequences. However, the marker E5/M12–600 showed polymorphism in RFLP analysis and was closely linked to the TGMS gene at a distance of 3.3 cM. This marker was subsequently mapped on chromosome 2 using doubled-haploid mapping populations derived from the crosses IR64×Azucena and CT9993×IR62666, available at IRRI, Philippines, and Texas Tech University, respectively. Linkage of microsatellite marker RM27 with the TGMS gene further confirmed its location on chromosome 2. The closest marker, E5/M12–600, was sequenced so that a PCR marker can be developed for the marker-assisted transfer of this gene to different genetic backgrounds. The new TGMS gene is tentatively designated as tms4(t). Received: 13 July 1999 / Accepted: 27 July 1999     

Gas exchange and chlorophyll fluorescence responses of <Emphasis Type="Italic">Pinus radiata</Emphasis> D. Don seedlings during and after several storage regimes and their effects on post-planting survival

Post-storage gas exchange parameters like CO₂ assimilation, stomatal conductance, transpiration, water use efficiency and intercellular CO₂ concentrations, together with several chlorophyll a fluorescence parameters: F_o, F_v, F_v/F_m, F_m/F_o and F_v/F_o were examined in radiata pine (Pinus radiata D. Don) seedlings that were stored for 1, 8 or 15 days at 4° or 10°C with or without soil around the roots. Results were analysed in relation to post-storage water potential and electrolyte leakage in order to forecast their vitality (root growth potential) following cold storage, and post-planting survival potential under optimal conditions. During storage at 4° and 10°C, photosynthesis was reduced, being more pronounced in bare-root seedlings than in seedlings with soil around the roots. The depletion of CO₂ assimilation seemed not to be solely a stomatal effect as effects on chloroplasts contributed to this photosynthetic inhibition. Thus, the fall in the ratios F_v/F_m, F_v/F_o and F_m/F_o indicated photochemical apparatus damage during storage. Photosynthetic rate was positively correlated with the root growth index and new root length showing that new root growth is dependent primarily on current photosynthesis. Pre-planting exposure of bare-root radiata pine seedlings to temperatures of 10°C for more than 24 h during transportation or storage is not recommended.     

Cytological investigation of male sterility in sorghum caused by a dominant mutation (<Emphasis Type="Italic">Ms</Emphasis><Subscript><Emphasis Type="Italic">tc</Emphasis></Subscript>) derived from tissue culture

Male sterility mutations are an important tool in the investigation of anther and pollen development and for obtaining hybrid seeds in plant breeding. Cytological analysis of microsporo- and microgameto-genesis in sorghum plants with dominant mutation of male sterility (Ms_tc) derived from tissue culture has been carried out. Using substitution backcrosses, this mutation was introduced first into the nuclear background of the fertile sorghum line SK-723 and from this line into Volzhskoe-4w (V-4w). The mechanism of Ms_tc action on anther and pollen development differed in different nuclear backgrounds. In SK-723, phenotypic expression of Ms_tc began before the beginning of meiosis, which resulted in degeneration of sporogenous tissue in some anthers and in significant disturbances of anther morphology. In microsporocytes that did not degenerate, the frequency of non-specific meiotic abnormalities characteristic of the fertile line SK-723 significantly increased. In addition, in the mutant plants, a number of specific meiotic abnormalities--almost complete desynapsis, and formation of syncytial structures--were observed, apparently the consequence of Ms_tc action. In mono- or bi-nucleate microspores, degenerative processes resulting in formation of empty or anomalously coloured pollen grains led to almost complete male sterility. In the V-4w nuclear background, changes in anther structure and meiotic disturbances were infrequent. The degenerative processes began at the uni- or binucleate microspore stage and resulted in formation of empty or abnormally coloured pollen grains, and in partial pollen sterility. Thus, the same nuclear male sterility-inducing mutation in different nuclear backgrounds affects different stages of pollen development.     

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.     

Characterization and identification of the candidate gene of rice thermo-sensitive genic male sterile gene <Emphasis Type="Italic">tms5</Emphasis> by mapping

Previous research has demonstrated that the thermo-sensitive genic male-sterile (TGMS) gene in rice was regulated by temperature. TGMS rice is important to hybrid rice production because the application of the TGMS system in two-line breeding is cost-effective, simple, efficient and overcomes the limitations of the cytoplasmic male sterility (CMS) system. AnnongS is the first discovered and deeply studied TGMS rice line in China. Previous studies have suggested that AnnongS-1 and Y58S, two derivative TGMS lines of AnnongS, were both controlled by a single recessive gene named tms5, which was genetically mapped on chromosome 2. In the current study, three populations (AnnongS-1 × Nanjing11, Y58S × Q611, and Y58S × Guanghui122) were developed to investigate the tms5 gene molecular map. Analysis of recombination events of sterile samples, utilizing 125 probes covering the tms5 region, suggested that the tms5 gene was physically mapped to a 19 kb DNA fragment between two markers, 4039-1 and 4039-2, located on the BAC clone AP004039. Following the construction of a physical map between the two markers, ONAC023, a member of the NAC (NAM-ATAF-CUC-related) gene family, was identified as the candidate of the tms5 gene.     

The greater seedling high-light tolerance of <Emphasis Type="Italic">Quercus robur</Emphasis> over <Emphasis Type="Italic">Fagus sylvatica </Emphasis>is linked to a greater physiological plasticity

The responses of Quercus robur (oak) and Fagus sylvatica (beech) seedlings to four different light environments (full, 50%, 40% and 15% sunlight) and to a rapid increase in irradiance were explored during the summer, after 2 years of growth in a forest nursery at Nancy (France). Significant differences between the two species were found for most variables. Phenotypic plasticity for morphological variables (root-shoot ratio, leaf size, leaf weight ratio) was higher in beech than in oak, while the reverse was true for anatomical (stomatal density, epidermis thickness, exchange surface area of the palisade parenchyma) and physiological (maximum photosynthetic rate, stomatal conductance, Rubisco activity) variables. Predawn photochemical efficiency (F_v/F_m) was higher in oak than in beech in all light environments except in 15% sunlight. F_v/F_m was significantly lower in 100% sunlight than in the other light environments in beech but not in oak. Maximum photosynthetic rates (A_max) increased with increasing light availability in the two species but they were always higher in oak than in beech. Oak exhibited higher Rubisco activity than beech in full sunlight. The transfer of shade-adapted seedlings to the open caused a decrease of F_v/F_m, which was larger for beech than for oak. Transferred oak but not beech plants recovered gradually to the control F_v/F_m values. The decreased chlorophyll content and the increased non-photochemical quenching observed in high-light beech seedlings were not enough to avoid photoinhibition. The results suggest that a greater tolerance of strong irradiance is linked to an enhanced physiological plasticity (variables related to photosynthesis), while shade tolerance relies on an enhanced plasticity in light-harvesting variables (crown morphology and chlorophyll content).     

Photosynthesis, photoinhibition, and nitrogen use efficiency in native and invasive tree ferns in Hawaii

Photosynthetic gas exchange, chlorophyll fluorescence, nitrogen use efficiency, and related leaf traits of native Hawaiian tree ferns in the genus Cibotium were compared with those of the invasive Australian tree fern Sphaeropteris cooperi in an attempt to explain the higher growth rates of S. cooperi in Hawaii. Comparisons were made between mature sporophytes growing in the sun (gap or forest edge) and in shady understories at four sites at three different elevations. The invasive tree fern had 12-13 cm greater height increase per year and approximately 5 times larger total leaf surface area per plant compared to the native tree ferns. The maximum rates of photosynthesis of S. cooperi in the sun and shade were significantly higher than those of the native Cibotium spp (for example, 11.2 and 7.1 µmol m^-2 s^-1, and 5.8 and 3.6 µmol m^-2 s^-1 respectively for the invasive and natives at low elevation). The instantaneous photosynthetic nitrogen use efficiency of the invasive tree fern was significantly higher than that of the native tree ferns, but when integrated over the life span of the frond the differences were not significant. The fronds of the invasive tree fern species had a significantly shorter life span than the native tree ferns (approximately 6 months and 12 months, respectively), and significantly higher nitrogen content per unit leaf mass. The native tree ferns growing in both sun and shade exhibited greater photoinhibition than the invasive tree fern after being experimentally subjected to high light levels. The native tree ferns recovered only 78% of their dark-acclimated quantum yield (F_v/F_m), while the invasive tree fern recovered 90% and 86% of its dark-acclimated F_v/F_m when growing in sun and shade, respectively. Overall, the invasive tree fern appears to be more efficient at capturing and utilizing light than the native Cibotium species, particularly in high-light environments such as those associated with high levels of disturbance.     

High-resolution mapping of the bolting gene <Emphasis Type="Italic">B</Emphasis> of sugar beet

Sugar beet (Beta vulgaris L.) is a biennial species. Shoot elongation (bolting) starts after a period of low temperature. The dominant allele of locus B causes early bolting without cold treatment. This allele is abundant in wild beets whereas cultivated beets carry the recessive allele. Fifteen AFLP markers, tightly linked to the bolting locus, have been identified using bulked segregant analysis. The F₂-population consisted of 2,134 individuals derived after selfing a single F₁-plant (Bb). In a first step, a linkage map was established with 249 markers based on 775 F₂-individuals with a coverage of 822.3 cM. The loci are dispersed over nine linkage groups corresponding to the haploid chromosome number of Beta species. Seventeen marker loci were placed at a distance less than 3.2 cM around the bolting gene. In a second step, four of those markers most closely linked to B were mapped with the entire F₂-population. Two of the markers were mapped flanking the B gene at distances of 0.14 and 0.23 cM. The other two markers were mapped at a distance of 0.5 cM from the gene. The tight linkage could be verified by testing 88 unrelated plants from a breeding program. The closely linked markers will enable breeders to select for the non-bolting character without laborious test crossings. Moreover, these markers are being used for map-based cloning of the bolting gene.     

Growth, photosynthesis and ozone uptake of young beech (Fagus sylvatica L.) in response to different ozone exposures

Beech seedlings (Fagus sylvatica L.) were exposed to episodes of O₃ in environmentally controlled growth chambers during one growing season. Three treatments were applied: charcoal-filtered air, charcoal-filtered air with the addition of 40 ppb O₃ for seven episodes of 5 days' duration (9000-1700 hours), and charcoal-filtered air with the addition of 100 ppb O₃ for seven episodes of 5 days' duration (9000-1700 hours). The accumulated exposure over a threshold of 40 ppb in the last treatment reached 13,911 ppb h. Throughout the growing season we measured growth as well as photosynthetic properties and related effects to external and calculated internal doses of O₃, using stomatal conductance (g_s) data. Growth, measured as diameter increment and biomass, was not significantly affected by the O₃ treatments. In the 100-ppb treatment, light-saturated CO₂ assimilation rates and chlorophyll content were significantly reduced, and the chlorophyll fluorescence parameter F_v/F_m was significantly reduced at times of high uptake rates and coincided with strong reductions of assimilation rates. O₃ uptake was lowered in the 100-ppb treatment due to reduced g_s. There was serious visible damage by the end of the exposure period in the 100-ppb treatment, while the treatment with 40 ppb O₃ did not seem to cause any significant changes.     

Tagging and mapping the thermo-sensitive genic male-sterile gene in rice (Oryza sativa L.) with molecular markers

The thermo-sensititve genic male-sterile (TGMS) gene in rice can alter fertility in response to temperature and is useful in the two-line system of hybrid rice production. However, little is known about the TGMS gene at the molecular level. The objective of this study was to identify molecular markers tightly linked with the TGMS gene and to map the gene onto a specific rice chromosome. Bulked segregant analysis of an F₂ population from 5460s (a TGMS mutant line) x Hong Wan 52 was used to identify RAPD markers linked to the rice TGMS gene. Four hundred RAPD primers were screened for polymorphisms between the parents and between two bulks representing fertile and sterile plants; of these, 4 primers produced polymorphic products. Most of the polymorphic fragments contained repetitive sequences. Only one singlecopy sequence fragment was found, a 1.2-kb fragment amplified by primer OPB-19 and subsequently named TGMS1.2. TGMS1.2 was mapped on chromosome 8 with a RIL population and confirmed by remapping with a DHL population. Segregation analysis using TGMS1.2 as a probe indicated that TGMS1.2 both consegregated and was lined with the TGMS gene in this population. It is located about 6.7 cM from the TGMS gene. As TGMS1.2 is linked to the TGMS gene, the TGMS gene must be located on chromosome 8.This research was supported by the Rockefeller Foundation and China National High-Tech Research and Development Program. The first author is a Rockefeller Career Fellow at Texas Tech University     

A genetic linkage map of water yam ( Dioscorea alata L.) based on AFLP markers and QTL analysis for anthracnose resistance

A genetic linkage map of the tetraploid water yam (Dioscorea alata L.) genome was constructed based on 469 co-dominantly scored amplified fragment length polymorphism (AFLP) markers segregating in an intraspecific F₁ cross. The F₁ was obtained by crossing two improved breeding lines, TDa 95/00328 as female parent and TDa 87/01091 as male parent. Since the mapping population was an F₁ cross between presumed heterozygous parents, marker segregation data from both parents were initially split into maternal and paternal data sets, and separate genetic linkage maps were constructed. Later, data analysis showed that this was not necessary and thus the combined markers from both parents were used to construct a genetic linkage map. The 469 markers were mapped on 20 linkage groups with a total map length of 1,233 cM and a mean marker spacing of 2.62 cM. The markers segregated like a diploid cross-pollinator population suggesting that the water yam genome is allo-tetraploid (2n = 4x = 40). QTL mapping revealed one AFLP marker E-14/M52-307 located on linkage group 2 that was associated with anthracnose resistance, explaining 10% of the total phenotypic variance. This map covers 65% of the yam genome and is the first linkage map reported for D. alata. The map provides a tool for further genetic analysis of traits of agronomic importance and for using marker-assisted selection in D. alata breeding programmes. QTL mapping opens new avenues for accumulating anthracnose resistance genes in preferred D. alata cultivars.