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     

Molecular mapping of the reverse thermo-sensitive genic male-sterile gene (rtms1) in rice

TGMS (thermo-sensitive genic male-sterile) rice is widely used in hybrid rice production. Because of a specific temperature requirement, it can be used only in a narrow rice-growing zone in Asia. A newly discovered reverse thermo-sensitive genic male-sterile line, J207S, has an opposite phynotype compared to the normal TGMS lines. J207S is completely sterile when the temperature is lower than 31°C. Thus, it can be widely used in a larger area. Genetic analysis indicated that the sterility of J207S was controlled by a single recessive gene which was first named as rtms1. An F₂ population from the cross between J207S and E921 was developed and used for molecular mapping of the rtms1 gene. The AFLP (amplified fragment length polymorphism) technique, combined with BSA (bulked segregant analysis), was used to screen markers linked to the target gene, and eight polymorphic AFLP loci were identified. Co-segregating analysis using the F₂ population showed that two of them, Rev1 and Rev7, were closely linked to the target gene with a recombinant rate of 3.8% and 7.7%, respectively. Both Rev1 and Rev7 were found to be single-copy sequences through Southern analysis. Rev1 was subsequently mapped on chromosome 10 with a doubled-haploid mapping populations derived from the cross CT9993 × IR62266 available at Texas Tech University. RM222 and RG257 were linked to Rev1 at a distance of 11.8 cM and 4.6 cM, respectively. Additional SSR markers from the rice map of Cornell University, RFLP markers from the map of RGP in Japan and the map of Texas Tech University were selected from the region surrounding Rev1 on chromosome 10 to conduct the fine-mapping of the rtms1 gene. Presently, rtms1 was mapped between RM239 and RG257 with genetic distance of 3.6 cM and 4.0 cM, respectively. The most-closely linked AFLP marker, Rev1, 4.2 cM from the rtms1 gene, was sequenced and converted into a SCAR (sequence characterized amplified region) marker which could facilitate marker-assisted selection of the rtms1 gene. Received: 2 November 2000 / Accepted: 21 November 2000     

Genetic analysis,molecular tagging and mapping of the thermo-sensitive genic male-sterile gene (wtms1) in wheat

A thermo-sensitive genic male-sterile (TGMS) wheat line ( Triticum aestivum L.) BNY-S was obtained from the spontaneous mutant of BNY-F. Its fertility was decided by the temperature during the differentiation stage of the spikelets. BNY-S was completely sterile when the temperature was lower than 10 degrees C during the differentiation stage of the spikelets, but fertile when the temperature was higher than 10 degrees C. Genetic analysis indicated that the sterility of BNY-S was controlled by a single recessive gene, which was named as wtms1. An F(2) population, consisting of 3,000 individuals from the cross between BNY-S and Lankao 52-24, was used for genetic analysis and statistical analysis of the TGMS and, out of them, 158 sterile and 93 fertile extremes were present for molecular tagging and mapping of the wtms1 gene. SSR (simple sequence repeat) and AFLP (amplified fragment length polymorphism) techniques combined with BSA (bulked segregant analysis) were used to screen markers linked to the target gene. As a result, wtms1 was preliminarily mapped on chromosome 2B according to SSR analysis. In AFLP analysis, 14 polymorphic AFLP loci were identified with a linkage relation to the wtms1 gene. Then linkage analysis using the F(2) population showed that three of them, E: AAG/M: CTA(163), E: AGG/M: CTC(220) and E: ACA/M: CTA(160), were linked to the wtms1 gene relatively close to a genetic distance of 6.9 cM, 6.9 cM and 13.9 cM, respectively. Finally, the wtms1 gene was mapped between the SSR marker Xgwm 374 and the AFLP marker E: AAG/M: CTA(163) with the distance of 4.8 cM and 6.9 cM, respectively. A partial linkage map was constructed according the SSR and AFLP data.     

Inheritance of seed colour and identification of RAPD and AFLP markers linked to the seed colour gene in rapeseed (Brassica napus L.)

In China Polima cytoplasmic male sterility (cms) is currently the most important hybrid system used for the breeding of hybrids. In an effort to develop yellow-seeded Polima cms restorer lines, we used yellow-seeded, doubled haploid (DH) line No.2127-17 as the gene source in crosses with two elite black-seeded Polima cms R lines, Hui5148-2 and 99Yu42, which originated from our breeding programme. The inheritance of seed colour was investigated in the F₂, BC₁ and F₁-derived DH progenies of the two crosses. Seed colour was found to be under the control of the maternal genotype and the yellow seed trait to be partially dominant over the black seed trait. Segregation analysis revealed a single gene locus for the partial dominance of yellow seed colour. Of 810 randomly amplified polymorphic DNA (RAPD) primers, 240 (29.6%) revealed polymorphisms between the parents. Of the 240 RAPD primers and 512 amplified fragment length polymorphism (AFLP) primer pairs, four RAPDs and 16 AFLP pairs showed polymorphisms between the bulks, with two RAPD and eight AFLP markers being identified in the vicinity of the seed-coat colour gene locus using a DH progeny population—derived from the cross Hui5148-2×No.2127-17—of 127 individuals in combination with the bulked segregant analysis strategy. Seven of these latter ten markers were linked to the allele for yellow seed, whereas the other three were linked to the allele for black seed. The seed-coat colour gene locus was bracketed by two tightly linked markers, EA02MG08 (2.4 cM) and S1129 (3.9 cM). The partial dominance and single gene control of the yellow seed-coat colour trait together with the available molecular markers will greatly facilitate the future breeding of yellow-seeded hybrid varieties.     

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 validation of multiple allele inheritance for dominant genic male sterility gene in Brassica napus L

Dominant genic male sterility (DGMS) has been playing an increasingly important role, not only as a tool for assisting in recurrent selection but also as an alternative approach for efficient production of hybrids. Previous studies indicate that fertility restoration of DGMS is the action of another unlinked dominant gene. Recently, through classical genetic analysis with various test populations we have verified that in a DGMS line 609AB the trait is inherited in a multiple allelic pattern. In this study, we applied molecular marker technology to provide further validation of the results. Eight amplified fragment length polymorphism (AFLP) markers tightly linked to the male sterility allele (Ms) were identified in a BC₁ population from a cross between 609A (a sterile plant in 609AB) and a temporary maintainer GS2467 as recurrent parent. Four out of the eight markers reproduced the same polymorphism in a larger BC₁ population generated with microspore-derived doubled haploid (DH) parents (S148 and S467). The two nearest AFLP markers SA12MG14 and P05MG15, flanking the Ms locus at respective distances of 0.3 centiMorgan (cM) and 1.6 cM, were converted into sequence characterized amplified region (SCAR) markers designated SC6 and SC9. Based on the sequence difference of the marker P05MG15 between S148 and a DH restorer line S103, we further developed a SCAR marker SC9f that is specific to the restorer allele (Mf). The map distance between SC9f and Mf was consistent with that between SC9 and Ms allele. Therefore, successful conversion of the marker tightly linked to Ms into a marker tightly linked to Mf suggested that the restoration for DGMS in 609AB is controlled by an allele at the Ms locus or a tightly linked gene (regarded as an allele in practical application). The Ms and Mf-specific markers developed here will facilitate the breeding for new elite homozygous sterile lines and allow further research on map-based cloning of the Ms gene.     

Genetic analysis and molecular mapping of a nuclear recessive male sterility gene, ms91(t), in rice.

Mutations that result in plant male sterility provide means not only to probe reproductive development but also to facilitate commercial heterosis application and hybrid seed production. In this study, we report a novel male sterility gene, ms91(t), in a spontaneous mutant line (SH38) from a Chinese rice cultivar (Oryza sativa subsp. japonica 'Jijing14'). The sterility of SH38 was studied by examining its progenies derived from crosses with 6 japonica cultivars. Corresponding F2 populations were obtained by selfing each of the 6 F1s and a backcross population was produced by crossing SH38 to the F1 of SH38 x C18. Our results revealed that SH38 has normal agronomic traits but produces no pollen grains. Segregations of male-sterile and male-fertile progenies in the F2 and backcross populations fit well with ratios of 3:1 and 1:1, respectively, indicating that ms91(t) is a single recessive gene. Amplified fragment length polymorphism (AFLP) analysis of SH38 and Jijing14 plants showed the presence of a unique band in SH38. Simple sequence repeat (SSR) analysis of the bulked and individual progenies of the F2 population of SH38 x C18 showed linkage of ms91(t) with the SSR marker RM5853 on chromosome 1. Subsequently, ms91(t) was fine-mapped to the interval between markers RM7075 (3.75 cM) and RM5638 (3.57 cM). Our results would facilitate the isolation of ms91(t) and male sterility in heterosis application.     

Molecular tagging and mapping of the erect panicle gene in rice

Erect panicle (EP) is one of the more important traits of the proposed ideotype of high-yielding rice. Several rice cultivars with the EP phenotype, which has been reported to be controlled by a dominant gene, have been successfully developed and released for commercial production in North China. To analyze the inheritance of the EP trait, we generated segregating F₂ and BC₁F₁ populations by crossing an EP-type variety, Liaojing 5, and a curved-panicle-type variety, Fengjin. Our results confirmed that a dominant gene controls the EP trait. Simple-sequence repeat (SSR) and bulked segregant analyses of the F₂ population revealed that the EP gene is located on chromosome 9, between two newly developed SSR markers, RM5833-11 and RM5686-23, at a genetic distance of 1.5 and 0.9 cM, respectively. Markers closer to the EP gene were developed by amplified fragment length polymorphism (AFLP) analysis with 128 AFLP primer combinations. Three AFLP markers were found to be linked to the EP gene, and the nearest marker, E-TA/M-CTC₂₀₀, was mapped to the same location as SSR marker RM5686-23, 1.5 cM from the EP gene. A local map around the EP gene comprising nine SSR and one AFLP marker was constructed. These markers will be useful for marker-assisted selection (MAS) for the EP trait in rice breeding programs.     

Identification of AFLP markers linked to the male fertility restorer gene of CMS (Moricandia arvensis) Brassica juncea and conversion to SCAR marker

We have developed a cytoplasmic male sterile (CMS) line of Brassica juncea through somatic hybridization with Moricandia arvensis and introgressed the fertility restorer gene into B. juncea. This fertility restorer locus is unique in that it is capable of restoring male fertility to two other alloplasmic CMS systems of B. juncea. As a first step toward cloning of this restorer gene we attempted molecular tagging of the Rf locus using the amplified fragment length polymorphism (AFLP) technique. A BC₁F₁ population segregating for male sterility/fertility was used for tagging using the bulk segregant analysis method. Out of 64 primer combinations tested in the bulks, 5 combinations gave polymorphic amplification patterns. Further testing of these primers in individual plants showed four amplicons associated with the male fertility trait. Polymorphic amplicons were cloned and used for designing SCAR primers. One of the SCAR primers generated amplicons mostly in the fertile plants. Linkage analysis using MAPMAKER showed two AFLP and one SCAR markers linked to the male fertility gene with a map distance ranging from 0.6 to 2.9 cM. All the markers are located on one side of the Rf locus.     

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.     

A bentazon and sulfonylurea sensitive mutant: breeding,genetics and potential application in seed production of hybrid rice

The use of a thermosensitive genic male sterility (TGMS) system in two-line hybrid rice breeding is affected greatly by the sterility instability of TGMS lines caused by temperature fluctuation beyond their critical temperatures for fertility reversion. To prevent seed production from self contamination, we have developed a system to secure seed purity using a herbicide-sensitive TGMS mutant, M8077S, obtained by radiation. Genetic analysis, using the F₁, F₂ and F₃ populations derived from this mutant and other normal varieties, revealed that bentazon lethality/sensitivity was controlled by a single recessive gene, which was named bel. The mutant can be killed at the seedling stage by bentazon at 300 mg/l or higher, a dosage that is safe for its F₁ hybrids and all other normal varieties. This mutant is also sensitive to all the tested sulfonylurea herbicides. Response of segregating plants to these two types of herbicide indicated that sulfonylurea sensitivity was also controlled by bel. By crossing this mutant with Pei-Ai 64S, an F₂ population was developed for genetic mapping. Surveying the two DNA pools from sensitive and non-sensitive F₂ plants identified four markers that were polymorphic between the pools. The putative linked markers were then confirmed with the F₂ population. The bel locus was located on chromosome 3, 7.1 cM from the closest microsatellite marker RM168. Phenotypic analysis indicated that the bel gene had no negative effect on agronomic traits in either a homozygous or heterozygous status. The mutant M8077S is valuable in the development of a TGMS breeding system for preventing impurity resulting from temperature fluctuation of the TGMS. Several two-line hybrid rice crosses using this system are under development.     

A high-density molecular map for ryegrass (Lolium perenne) using AFLP markers

AFLP markers have been successfully employed for the development of a high-density linkage map of ryegrass (Lolium perenne L.) using a progeny set of 95 plants from a testcross involving a doubled-haploid tester. This genetic map covered 930 cM in seven linkage groups and was based on 463 amplified fragment length polymorphism (AFLP) markers using 17 primer pairs, three isozymes and five EST markers. The average density of markers was approximately 1 per 2.0 cM. However, strong clustering of AFLP markers was observed at putative centromeric regions. Around these regions, 272 markers covered about 137 cM whereas the remaining 199 markers covered approximately 793 cM. Most genetic distances between consecutive pairs of markers were smaller than 20 cM except for five gaps on groups A, C, D, F and G. A skeletal map with a uniform distribution of markers can be extracted from this high-density map, and can be applied to detect and map QTLs. We report here the application of AFLP markers to genome mapping, in Lolium as a prelude to quantitative trait locus (QTL) identification for diverse agronomic traits in ryegrass and for marker-assisted plant breeding. Received: 4 November 1998 / Accepted:15 March 1999     

Fine mapping of the rice thermo-sensitive genic male-sterile gene <Emphasis Type="Italic">tms5</Emphasis>

AnnongS-1, a thermo-sensitive genic male-sterile (TGMS) rice line, has a new TGMS gene. Genetic analysis indicated that the sterility of AnnongS-1 was controlled by a single resessive gene named tms5. In our previous studies based on an F₂ population from the cross between AnnongS-1 and Nanjing11, tms5 was mapped on chromosome 2. Recently, a RIL (recombinant inbred line) population from the same cross was developed and used for the fine mapping of the tms5 gene. Molecular marker techniques combined with BSA (bulked segregant analysis) were used. As a result, two AFLP markers (AF10, AF8), one RAPD marker (RA4), one STS marker (C365-1), one CAPs marker (G227-1) and four SSR markers (RM279, RM492, RM327, RM324) were found to be closely linked to tms5 gene. The DNA sequences of the RFLP marker of C365 and G227 were found in GenBank, and on the basis of these sequences, many primers were designed to amplify the two parents and their RIL population plants. Finally, the tms5 gene was mapped between STS marker C365-1 and CAPs marker G227-1 at a distance of 1.04 cM from C365-1 and 2.08 cM from G227-1.Communicated by H.F. LinskensY.G. Wang and Q.H. Xing contributed equally to this contribution.     

Molecular markers linked to stem rot resistance in rice

Stem rot (Sclerotium oryzae) is an important disease constraint in Californian rice production. Measurement of resistance is laborious, and the low heritability of the trait limits the effectiveness of selection in breeding programs. Molecular markers linked to the trait would therefore provide a superior selection screen to assist in transferring resistance into improved cultivars. The genetics of resistance to stem rot was studied in the germplasm line 87-Y-550 (PI566666), which inherited its resistance from the wild species Oryza rufipogon. Four crosses of 87-Y-550 with susceptible lines were made and recombinant inbred lines of only the most-resistant and most-susceptible progeny within each cross were advanced for late-generation testing. Approximately 900 AFLP (amplified fragment length polymorphism) primer combinations were applied to resistant and susceptible bulks within each cross. One AFLP marker showed significant association with stem rot resistance and accounted for approximately 45.0% of the phenotypic variation in 59 progenies. This marker was mapped on rice chromosome 2 between the RFLP markers RZ166 and RG139 by using F₂-reference population information. The accuracy of AFLP marker mapping was validated by size and sequence comparison of AFLP bands from 87-Y-550 and the reference population. With the strategy of selective genotyping combined with a parental survey, two microsatellite markers, RM232 and RM251, on chromosome 3 were also found associated with stem rot resistance and accounted for 41.1% and 37.9% of the phenotypic variation, respectively. The multiple linear regression model included TAA/GTA167 on chromosome 2 and RM232 on chromosome 3 and cumulatively explained 49.3% of total variation. The molecular markers linked to stem rot resistance should facilitate selection for this recalcitrant trait in rice breeding programs by eliminating the need for early generation screening. Received: 27 March 2000 / Accepted: 4 June 2000     

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.     

Primary genetic linkage maps of the ascidian, Ciona intestinalis

For whole-genome analysis in a basal chordate (protochordate), we used F1 pseudo-testcross mapping strategy and amplified fragment length polymorphism (AFLP) markers to construct primary linkage maps of the ascidian tunicate Ciona intestinalis. Two genetic maps consisted of 14 linkage groups, in agreement with the haploid chromosome number, and contained 276 and 125 AFLP loci derived from crosses between British and Neapolitan individuals. The two maps covered 4218.9 and 2086.9 cM, respectively, with an average marker interval of 16.1 and 18.9 cM. We observed a high recombinant ratio, ranging from 25 to 49 kb/cM, which can explain the high degree of polymorphism in this species. Some AFLP markers were converted to sequence tagged sites (STSs) by sequence determination, in order to create anchor markers for the fragmental physical map. Our recombination tools provide basic knowledge of genetic status and whole genome organization, and genetic markers to assist positional cloning in C. intestinalis.     

Pollen viability restoration in a <Emphasis Type="Italic">Coffea canephora</Emphasis> P. and <Emphasis Type="Italic">C. heterocalyx</Emphasis> Stoffelen backcross. QTL identification for marker-assisted selection

Male fertility of interspecific hybrids was analysed in one F1 and two backcrossed progenies originating from a cross between Coffea canephora and Coffea heterocalyx. Male fertility was tested using pollen stainability with acetic carmine. The results showed a marked decline in fertility at the F1 level, and fertility was almost fully restored after two backcrosses. The computed broad-sense heritability represented 47% of the variance. Quantitative trait loci (QTLs) locations and effects on pollen viability were estimated using an amplified fragment length polymorphism (AFLP) genetic linkage map constructed in the segregating BC1 population. Three significant QTLs (LOD>3 and p < 0.001 by ANOVA) were detected for pollen viability, two of which were responsible for the bimodal distribution of pollen viability in the segregating population. One QTL was involved in fertility variations among fertile BC1 plants. Fertility inheritance is discussed in relation with previously demonstrated chromosomal sterility in Coffea hybrids and the effect of detected QTLs. The potential use of genetic markers to overcome sterility in interspecific hybrids is also discussed.     

Construction of a high-resolution linkage map of Rfd1, a restorer-of-fertility locus for cytoplasmic male sterility conferred by DCGMS cytoplasm in radish (Raphanus sativus L.) using synteny between radish and Arabidopsis genomes

Cytoplasmic male sterility caused by Dongbu cytoplasmic and genic male-sterility (DCGMS) cytoplasm and its nuclear restorer-of-fertility locus (Rfd1) with a linked molecular marker (A137) have been reported in radish (Raphanus sativus L.). To construct a linkage map of the Rfd1 locus, linked amplified fragment length polymorphism (AFLP) markers were screened using bulked segregant analysis. A 220-bp linked AFLP fragment sequence from radish showed homology with an Arabidopsis coding sequence. Using this Arabidopsis gene sequence, a simple PCR marker (A220) was developed. The A137 and A220 markers flanked the Rfd1 locus. Two homologous Arabidopsis genes with both marker sequences were positioned on Arabidopsis chromosome-3 with an interval of 2.4 Mb. To integrate the Rfd1 locus into a previously reported expressed sequence tag (EST)-simple sequence repeat (SSR) linkage map, the radish EST sequences located in three syntenic blocks within the 2.4-Mb interval were used to develop single nucleotide polymorphism (SNP) markers for tagging each block. The SNP marker in linkage group-2 co-segregated with male fertility in an F(2) population. Using radish ESTs positioned in linkage group-2, five intron length polymorphism (ILP) markers and one cleaved amplified polymorphic sequence (CAPS) marker were developed and used to construct a linkage map of the Rfd1 locus. Two closely linked markers delimited the Rfd1 locus within a 985-kb interval of Arabidopsis chromosome-3. Synteny between the radish and Arabidopsis genomes in the 985-kb interval were used to develop three ILP and three CAPS markers. Two ILP markers further delimited the Rfd1 locus to a 220-kb interval of Arabidopsis chromosome-3.     

Identification of a deletion in <Emphasis Type="Italic">tms2</Emphasis> and development of gene-based markers for selection

Rice is one of the most important food crops. The temperature-sensitive genic male sterility (TGMS) system provides a great potential for improving food production by hybrids. The use of TGMS system is simple, inexpensive, effective, and eliminates the limitations of the conventional three-line system. A rice gene, tms2, generated by irradiation of a japonica variety has been reported to control TGMS in several rice lines. Previous studies reported genetic markers linked to this gene, and the gene was transferred to an aromatic Thai cultivar. Using information obtained from published databases, we located positions of the reported genetic markers flanking the gene in rice genomic sequences, and developed gene-based markers located inside the flanking markers for polymorphism detection. We found that inbred indica tms2 mutant plants contain about 1 Mb of japonica DNA, in which at least 70 kb was deleted. Using RT-PCR for expression analysis, four genes out of seven genes annotated as expressed proteins located inside the deletion showed expression in panicles. These genes could be responsible for TGMS phenotypes of tms2. In addition, we developed gene-based markers flanking and inside the deletion for selecting the tms2 gene in breeding populations. By genotyping 102 diverse rice lines including 38 Thai rice lines, 5 species of wild rice, and 59 exotic rice lines including TGMS lines and cultivars with desirable traits, a gene-based marker located inside the deletion and one flanking marker were shown to be highly specific for the tms2 mutant.     

Delimitation of the fertility restorer locus Rfk1 to a 43-kb contig in Kosena radish (Raphanus sativus L.)

We are pursuing a positional cloning strategy to isolate the fertility restoration gene Rfk1 from radish. Random polymorphic DNA-sequence-tagged site (RAPD-STS) markers tightly linked to the gene in radish were isolated, and a RAPD map surrounding the Rfk1 locus was constructed. We surveyed 948 F2 plants with adjacent RAPD-STS markers to isolate recombinants for bulk segregant analysis. This analysis was effective in isolating tightly linked amplification fragment length polymorphism (AFLP) markers surrounding the gene of interest. Ten tightly linked AFLP markers were obtained and used to construct a high-resolution map of the region. The closest AFLP-STS markers flanking Rfk1 were 0.1 cM and 0.2 cM away. Using the four adjacent AFLP markers, we screened lambda and cosmid libraries. The lambda and cosmid clones were aligned by examination of end sequences and restriction fragment length polymorphism (RFLP) patterns for each clone, and by hybridization to the DNA isolated from recombinants. Finally, we constructed a 198-kb contig encompassing the Rfk1 gene and comprising 20 lambda and two cosmid clones. By analysis of the breakpoints in recombinants with the rfk1/rfk1 or Rfk1/- genotype, the Rfk1 locus could be assigned to a 43-kb region comprising four lambda clones and one cosmid clone. This pinpoint localization in the radish genome has made it possible for us to identify the gene by sequence analysis and genetic transformation of cytoplasmic male-sterile Brassica napus plants.