Published in Crop Sci. 43:2267-2271 (2003).
© 2003 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY
Microsatellite Markers Flanking the tms2 Gene Facilitated Tropical TGMS Rice Line Development
M. T. Lopez*,a,
T. Toojindac,
A. Vanavichitb and
S. Tragoonrungc
a Bangkok Seed Industries Co. Ltd., Soi Yenchit, Chand Road Sathorn Bangkok, 10120 Thailand
b Kasetsart University, Kamphaengsaen Campus, Nakorn Pathom 73140 Thailand
c BIOTEC, National Center for Genetic Engineering and Biotechnology, Kasetsart University, Kamphaengsaen Campus, Nakorn Pathom 73140 Thailand
* Corresponding author (milagroslopez{at}lycos.com).
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ABSTRACT
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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.
Abbreviations: cM, centimorgan • CMS, cytoplasmic male sterility • KDML 105, Khaw Dawk Mali 105 • LOD, log likelihood ratio • RFLP, restriction fragment length polymorphism • TGMS, thermosensitive genetic male sterility
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INTRODUCTION
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HYBRID RICE TECHNOLOGY has tremendously improved rice productivity as effectively demonstrated in China and other Asian countries. To date, the CMS or three-line method is effective and widely used in producing hybrid rice. In the tropics, use of TGMS has been effective in developing hybrids and has shown prospects in increasing the efficiency of hybrid rice breeding (Lu et al., 1998; Lopez and Virmani, 2000). Developing TGMS lines is one of the basic steps in obtaining superior two-line rice hybrids. However, some problems are being encountered by breeders in handling the breeding lines because of the inherent nature of the TGMS trait and the fluctuating temperatures affecting its expression. Plant breeders at Kasetsart University have been working on the development of an efficient breeding program to generate suitable TGMS lines. With the availability of high-density rice molecular maps and molecular markers, molecular marker-aided breeding was considered a promising approach to solve these problems.
Genetic studies have shown that a recessive nuclear gene controlled the TGMS trait in rice. At present, five single recessive TGMS genes located on different chromosomes have been reported (Sun et al., 1989; Maruyama et al., 1991; Wang et al., 1995; Subudhi et al., 1997; Dong et al., 2000; Reddy et al., 2000). The tms2 gene was mapped on chromosome 7 by means of restriction fragment length polymorphism (RFLP) markers (Yamaguchi et al., 1997). This study took advantage of the known information regarding chromosomal location of the tms2 gene and the availability of many mapped microsatellite markers in rice (Temnykh et al., 2000). In this study, microsatellite markers associated with tms2 were determined and immediately used in selection in the TGMS line breeding program. Microsatellite markers associated with the tms2 gene were used to rapidly screen for its presence in the breeding population, thus facilitating the development of promising TGMS lines with aromatic background.
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MATERIALS AND METHODS
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Plant Materials and Phenotyping
A segregating F2 population consisting of 157 plants derived from the cross Norin PL12/KDML105 was used as mapping population to find microsatellite markers associated with tms2. Norin PL12 is the original source of the tms2 gene, while KDML105 is a popular Thai aromatic rice cultivar. In the backcross breeding program, two indica TGMS lines derived from Norin PL12 (IR68942-1-6-13-B-4 and IR68935-16-6-27), and two aromatic Thai rice cultivars, KDML105 and BKNA6-18-3-2 were used. The three backcrosses were IR68942-1-6-13-B-4/KDML105//KDML105, IR68942-1-6-13-B-4/BKNA6-18-3-2//BKNA6-18-3-2, and IR68935-16-6-27/KDML105//KDML105.
The right time to sow the seeds of the mapping population to observe segregation of the TGMS trait was inferred from the 25-yr (1973–1997) mean monthly maximum and minimum temperature data at Kasetsart University, Kamphaengsaen campus (data not shown). The mapping population was sown in the first week of December 1998. Some panicles were cut and fertilizer was applied regularly to induce continuous production of new tillers and panicles until the prevailing temperature was above 30°C in March. Observation of the pollen sterility or fertility of each F2 plant was taken twice, first, from the panicle that developed when maximum temperature was lower than 30°C 1 to 2 wk after panicle initiation, and the second, from the panicle that developed under high-temperature condition. Anthers of each F2 plant were observed visually. Microscopic observation of pollen of each F2 plant was also done to confirm visual observation of the anther color. Five to 10 flowers that would open the following day were collected and stored in 70% (v/v) ethanol until the time for observation. Pollen sterility was determined by staining pollen grains with 1% (w/v) IKI solution. Pollen grains were classified on the basis of their shape and extent of staining. The unstained withered or spherical pollen grains and the lightly stained round pollen grains were classified as sterile. The fertile pollen grains were black and round. Plants were classified on the basis of the extent of pollen sterility as follows: pollen-free (no pollen grains), completely sterile (100%), sterile (91–99%), partially sterile (71–90%), partially fertile (31–70%), and fertile (0–30%) (Virmani et al., 1997).
DNA Extraction and Microsatellite Analysis
Total genomic DNA was prepared from fresh-frozen leaf tissue of Norin PL 12, IR68942-1-6-13-B-4, IR68935-16-6-27, KDML105, BKNA6-18-3-2, the mapping population, and from the three backcross populations by the cetyl-trimethyl-ammonium bromide (CTAB) method by Murray and Thompson (1980) with some modifications. Enough ground leaf tissue to fill one-half of a 1.5-mL tube was sufficient to obtain enough DNA for microsatellite analyses.
Four microsatellite markers (RM2, RM10, RM11, and RM214) on chromosome 7 in the vicinity of tms2 and showing polymorphism between the two parents were used in genotyping the 157 F2 plants. The RM2, RM10, and RM11 markers were developed by Panaud et al. (1996), and the RM214 marker was developed by Chen et al. (1997). Total genomic DNA (5 ng) was used as template for PCR amplification. The PCR conditions were as follows: a hot start of 94°C for 30 s; 35 cycles of 1 min at 94°C denaturing temperature; 55°C annealing for 30 s and 72°C for extension for 1 min, and a 5-min final extension at 72°C. Polymorphism was analyzed by running PCR products in 4.5% (w/v) denaturing polyacrylamide gels (PAGE) at 80 to 100 V followed by silver staining. The PCR amplification products on the single polyacrylamide gel were loaded sequentially in genotyping all the individuals in the F2 population. Microsatellite markers were mapped by Mapmaker Version 3.0 (Lander et al., 1987). The maximum-likelihood map order for markers was determined with LOD score threshold of 3.0. Linkage analysis for the markers and the tms2 gene was conducted by MQTL (Tinker and Mather, 1995).
Molecular Marker-Aided Selection
The tms2 gene was previously transferred from Norin PL12 to the IRRI breeding lines designated as IR68935-16-6-27 and IR68942-1-6-13-B-4 (IRRI, 1997). These two TGMS lines were used as the tms2 gene sources to develop TGMS lines with aromatic background. Two aromatic Thai rice cultivars, KDML 105 and BKN A6-18-3-2, were used as recipient parents. The following backcrosses were made: IR68942-1-6-13-B-4/KDML105//KDML105, IR68942-1-6-13-B-4/BKNA6-18-3-2//BKNA6-18-3-2, and IR68935-16-6-27/KDML105//KDML105, and advanced up to the BC3 generation. In this breeding program, plants with phenotypic characters similar to the recurrent parent were selected for backcrossing. The accuracy of selection based on molecular markers was tested by growing 30 to 45 backcross F2 plants in the field when the temperature was above 30°C. BC3F1 plants were planted in the greenhouse to produce the BC3F2 seeds. Among the BC3F2, plants homozygous for the tms2 gene were identified at vegetative stage by means of the microsatellite markers. These identified plants were grown in the field until panicle initiation stage. These were uprooted and divided into two. One-half remained in the field and the other half was planted in pots. The potted plants were grown in an artificially illuminated air-conditioned room with an average 24°C diurnal temperature. Plants that produced seeds under low temperature condition but showed sterility under high temperature were selected. Seeds were harvested and planted for further evaluation and for generation advancement.
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RESULTS AND DISCUSSION
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Segregation of tms2 in the F2 population
The data on the segregation of fertile and sterile plants based on anther and pollen observations of the mapping population were taken from the late panicles that developed in March to April 1999. The 157 F2 plants segregated to 121 pollen-fertile and 36 pollen-sterile plants. The Chi-square test indicated that the population segregated in the ratio of three fertile plants to one sterile plant. This result confirmed that the TGMS trait is governed by a single recessive gene (Yang et al., 1992, Maruyama et al., 1991, Borkakati and Virmani, 1996; Dong et al., 2000, Reddy et al., 2000).
The early panicles of the mapping population that developed and flowered in mid-February to March showed partial to complete fertility. All plants had yellow anthers. Fertile pollen grains were round and stained black with I2KI when observed under the microscope. The 61 plants were partially fertile (31–70% pollen sterility) and 96 plants were fertile (0–30% pollen sterility). Exposure of the mapping population to fertility-inducing temperatures at the critical stage (15–25 d before heading or 5–15 d after panicle initiation) in January through February 1999 (30.8/19.0°C) induced all plants to produce fertile panicles. Segregation was observed in late panicles in this mapping population because the prevailing mean maximum/minimum temperatures in March to April 1999 were 35.6/23.4 to 37.5/24.6°C that resulted in the reversion of the fertile plants to sterile plants. The sterile plants had either unstained withered or spherical sterile pollen grains, or had pollen-free anthers. Pollen sterility data were used because results at IRRI farm over the years have shown that TGMS lines derived from Norin PL12 showed complete pollen sterility at temperatures above 30°C and 65.4 to 85.8% spikelet fertility at temperature under 30°C (Lu et al., 1998). The reversion of the fertile plants to sterile plants under high temperature in the critical stage indicated the presence of tms2 gene. Through this strategy, the segregation for pollen-fertile and pollen-sterile plants in the population, and the conversion from fertility to sterility of TGMS plants was observed. The result was similar to that report earlier (Lopez and Virmani, 2000).
Microsatellite Markers Flanking the tms2 Locus
Linkage analysis of the data indicated that RM11 and RM2 flank the tms2 locus on chromosome 7. RM11 was approximately 5 cM from tms2, while RM2 was approximately 16 cM away. In the F2 mapping population, 97% of the pollen-sterile plants were homozygous for tms2 (Norin PL12 allele) as determined by RM11. The pollen-fertile plants were either homozygous (98%) for the KDML105 allele, or heterozygous (95%) displaying both bands of KDML105 and Norin PL12 (Table 1, Fig. 1). As determined by RM2, 24 out of 26 (92%) sterile plants were homozygous for tms2. Among the fertile plants, 95% were homozygous and 87% were heterozygous for the KDML105 allele. These results indicated that both markers can be used to separate sterile from fertile plants. Although RM11 and RM2 markers were not very close to tms2 compare with the RFLP marker R634A (0.2 cM), as previously reported in Japan (Yamaguchi et al., 1997), the microsatellite marker, which is easier to use than the RFLP marker, showed high enough accuracy in separating the sterile from fertile plants in the breeding program.
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Table 1. Number of fertile and sterile plants among the F2 population of the cross Norin PL12/KDML 105 classified according to RM2 and RM11 microsatellite markers.
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Fig. 1. The portion of the polyacrylamide gel showing bands of RM2 and RM11 microsatellite markers of Norin PL12 and KDML105, and the segregation pattern of both markers and the corresponding phenotype (F = fertile, S = sterile) among 157 F2 plants of the Norin PL12/KDML105 cross.
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Selecting the TGMS Trait by RM11 and RM2 Markers
Microsatellite markers RM2 and RM11 were used to identify BC2F1 plants with the tms2 allele. The banding patterns of the BC2F1 individuals could be classified into those homozygous for KDML105 or BKNA6-18-3-2, and heterozygous displaying both bands of KDML105 or BKN A6-18-3-2 and IR68942-1-6-13-B-4 or IR68935-16-6-27 using RM11 and RM2 markers. To test the accuracy of selection for tms2 by means of RM2 and RM11 markers, the 213 BC2F2 were planted under the maximum temperature higher than 30°C. Of these plants, 69 were segregating for fertile and sterile plants, and 144 showed no segregation. Among the 69 segregating BC2F2 populations, 65 populations (94%) were progenies of heterozygous BC2F1 for RM2, and 41 (59%) were progenies of heterozygous BC2F1 for RM11 (Table 2). Among the 144 non-segregating BC2F2 populations, 92 and 89% were progenies of homozygous BC2F1 that had bands of the fertile parents (KDML105 or BKN A6-18-3-2) for RM2 and RM11, respectively. The results showed that both markers can be used to select for either homozygous or heterozygous plants by different cross combinations. Further analysis of the DNA of the 207 BC2F2 sterile plants showed that they were homozygous either in RM11, RM2, or both markers (Table 3). Among the BC2F2 sterile plants, 95% were homozygous for RM2, and 69% were homozygous for RM11. There were 67% of sterile plants that were homozygous for both markers. The results showed that fertile plants (Tmstms) possessing tms2 and homozygous sterile (tmstms) plants could be selected among other plants even in seedling stage and without exposing them to favorable environmental conditions for the expression of the TGMS gene.
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Table 2. Number of BC2F2 populations derived from 213 BC2F1 plants for genotypes at RM2 and RM11 for the backcross combinations: IR68942-1-6-13-B-4/KDML105//KDML105, IR68942-1-6-13-B-4/BKNA6-18-3-2//BKNA6-18-3-2, and IR68935-16-6-27/KDML105//KDML105.
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Table 3. Number of plants (%) and their genotypic profiles at either RM2, RM11 or both markers of 207 suspected TGMS plants selected from three rice backcross populations IR68942-1-6-13-B-4/KDML105//KDML105, IR68942-1-6-13-B-4/BKNA6-18-3-2//BKNA6-18-3-2, and IR68935-16-6-27/KDML105//KDML105.
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Performance of TGMS Lines in Different Temperature Regimes
The TGMS lines IR68935-16-6-27 and IR68942-1-6-13-B-4 showed over 70% spikelet fertility when exposed to 22 to 25°C diurnal temperature. However, when exposed to maximum/minimum temperatures of 35.5/23.1°C at the critical stage (1–2 wk after panicle initiation), they showed complete male sterility with either no pollen or 100% sterile pollen (Table 4). The results indicated that these TGMS lines are good TGMS gene sources.
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Table 4. Pollen and spikelet fertility, and characteristics of new TGMS lines and the parental lines evaluated at Kasetsart University, Kamphaengsaen campus, Nakorn Pathom, Thailand, in different seeding dates (December 1998, January 2000, and December 2001).
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The BC3F3 plants produced higher than 70% spikelet fertility because they were exposed to fertility-inducing temperature at the critical stage in January to February 2001. Late tillers of these plants showed complete spikelet sterility and complete pollen sterility when exposed to maximum/minimum temperatures of 32.6/23.5°C in March 2001. Three TGMS lines derived from the three backcrosses with acceptable spikelet fertility and agronomic characteristics were selected. The new TGMS lines were comparable to IR68942-1-6-13-B-4 and IR68935-16-6-27 in terms of fertility–sterility expression and adaptability to tropical conditions. Seed characteristics of these new TGMS lines were comparable to the Thai cultivars (Table 4). These results indicated that these lines have inherited TGMS trait from IR68942-1-6-13-B-4 and IR68935-16-6-27 and inherited seed characteristics of the Thai cultivars. These lines are being used as the new tms2 gene source to generate new TGMS lines for developing two-line tropical rice hybrids with good grain qualities.
The feasibility of marker-assisted selection for tms2 has been demonstrated in this study. The readily available mapped microsatellite markers in rice linked to tms2 have facilitated the development of tropical TGMS lines with aromatic Thai cultivar background. A large number of breeding lines can be handled by means of the microsatellite markers in the TGMS breeding program. The genotypes of 192 to 288 plants can be analyzed in a single polyacrylamide gel glass plate with 96-well combs that could be loaded two to three times. In generating advanced lines, the use of these markers can reduce the experimental area and effort expended to select lines segregating for sterility. These markers can be used to determine the genotype of the seedlings before transplanting to select heterozygous-fertile (Tmstms) plants and homozygous-sterile (tmstms) plants. Since only the heterozygous-fertile plants are expected to segregate for sterility under the maximum temperature higher than 30°C, the homozygous-fertile (TmsTms) plants can be discarded before transplanting. Transplanting homozygous-sterile lines in higher altitude areas with maximum temperature below 30°C to get self seeds until all other traits are fixed can reduce time to develop fix TGMS lines. These markers can determine the seedlings possessing tms2, so planting of breeding materials can be done any time of the year, because exposing them to a suitable temperature at the critical stage to allow TGMS gene expression will not be necessary. The results indicated that molecular marker-aided TGMS line breeding facilitated development of TGMS lines with aromatic Thai background.
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ACKNOWLEDGMENTS
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We thank Dr. Sant Singh Virmani of the Plant Breeding, Genetics and Biochemistry Division, IRRI, Philippines for providing the seeds of TGMS lines used in this study. The financial support of the Rockefeller Foundation is gratefully acknowledged. The technical assistance of the researchers at the DNA Technology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), Kasetsart University, Kamphaengsaen Campus, Nakorn Pathom 73140 Thailand is gratefully appreciated.
Received for publication June 14, 2002.
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