Single and multiple resistance QTL delay symptom appearance and slow down root colonization by Aphanomyces euteiches in pea near isogenic lines.

BACKGROUND: Understanding the effects of resistance QTL on pathogen development cycle is an important issue for the creation of QTL combination strategies to durably increase disease resistance in plants. The oomycete pathogen Aphanomyces euteiches, causing root rot disease, is one of the major factors limiting the pea crop in the main producing countries. No commercial resistant varieties are currently available in Europe. Resistance alleles at seven main QTL were recently identified and introgressed into pea agronomic lines, resulting in the creation of Near Isogenic Lines (NILs) at the QTL. This study aimed to determine the effect of main A. euteiches resistance QTL in NILs on different steps of the pathogen life cycle. RESULTS: NILs carrying resistance alleles at main QTL in susceptible genetic backgrounds were evaluated in a destructive test under controlled conditions. The development of root rot disease severity and pathogen DNA levels in the roots was measured during ten days after inoculation. Significant effects of several resistance alleles at the two major QTL Ae-Ps7.6 and Ae-Ps4.5 were observed on symptom appearance and root colonization by A. euteiches. Some resistance alleles at three other minor-effect QTL (Ae-Ps2.2, Ae-Ps3.1 and Ae-Ps5.1) significantly decreased root colonization. The combination of resistance alleles at two or three QTL including the major QTL Ae-Ps7.6 (Ae-Ps5.1/Ae-Ps7.6 or Ae-Ps2.2/Ae-Ps3.1/Ae-Ps7.6) had an increased effect on delaying symptom appearance and/or slowing down root colonization by A. euteiches and on plant resistance levels, compared to the effects of individual or no resistance alleles. CONCLUSIONS: This study demonstrated the effects of single or multiple resistance QTL on delaying symptom appearance and/or slowing down colonization by A. euteiches in pea roots, using original plant material and a precise pathogen quantification method. Our findings suggest that single resistance QTL can act on multiple or specific steps of the disease development cycle and that their actions could be pyramided to increase partial resistance in future pea varieties. Further studies are needed to investigate QTL effects on different steps of the pathogen life cycle, as well as the efficiency and durability of pyramiding strategies using QTL which appear to act on the same stage of the pathogen cycle.

Identification of environmentally stable QTL for resistance against Leptosphaeria maculans in oilseed rape (Brassica napus).

KEY MESSAGE: Six stable QTL for resistance against L. maculans (phoma stem canker) have been identified by QTL × environment interaction analysis using data from five winter oilseed rape field experiments. Phoma stem canker, caused by Leptosphaeria maculans, is a disease of worldwide importance on oilseed rape (Brassica napus). Quantitative trait loci (QTL)-mediated resistance against L. maculans in B. napus is considered to be race non-specific and potentially durable. Identification and evaluation of QTL for resistance to L. maculans is important for breeding oilseed rape cultivars with durable resistance. An oilseed rape mapping population was used to detect QTL for resistance against L. maculans in five winter oilseed rape field experiments under different environments. A total of 17 QTL involved in 'field' quantitative resistance against L. maculans were detected and collectively explained 51% of the phenotypic variation. The number of QTL detected in each experiment ranged from two to nine and individual QTL explained 2-25% of the phenotypic variation. QTL × environment interaction analysis suggested that six of these QTL were less sensitive to environmental factors, so they were considered to be stable QTL. Markers linked to these stable QTL will be valuable for selection to breed for effective resistance against L. maculans in different environments, which will contribute to sustainable management of the disease.

Validation of QTL for resistance to Aphanomyces euteiches in different pea genetic backgrounds using near-isogenic lines.

KEY MESSAGE: Marker-assisted backcrossing was used to generate pea NILs carrying individual or combined resistance alleles at main Aphanomyces resistance QTL. The effects of several QTL were successfully validated depending on genetic backgrounds. Quantitative trait loci (QTL) validation is an important and often overlooked step before subsequent research in QTL cloning or marker-assisted breeding for disease resistance in plants. Validation of QTL controlling partial resistance to Aphanomyces root rot, one of the most damaging diseases of pea worldwide, is of major interest for the future development of resistant varieties. The aim of this study was to validate, in different genetic backgrounds, the effects of various resistance alleles at seven main resistance QTL recently identified. Five backcross-assisted selection programs were developed. In each, resistance alleles at one to three of the seven main Aphanomyces resistance QTL were transferred into three genetic backgrounds, including two agronomically important spring (Eden) and winter (Isard) pea cultivars. The subsequent near-isogenic lines (NILs) were evaluated for resistance to two reference strains of the main A. euteiches pathotypes under controlled conditions. The NILs carrying resistance alleles at the major-effect QTL Ae-Ps4.5 and Ae-Ps7.6, either individually or in combination with resistance alleles at other QTL, showed significantly reduced disease severity compared to NILs without resistance alleles. Resistance alleles at some minor-effect QTL, especially Ae-Ps2.2 and Ae-Ps5.1, were also validated for their individual or combined effects on resistance. QTL × genetic background interactions were observed, mainly for QTL Ae-Ps7.6, the effect of which increased in the winter cultivar Isard. The pea NILs are a novel and valuable resource for further understanding the mechanisms underlying QTL and their integration in breeding programs.

Quantitative resistance affects the speed of frequency increase but not the diversity of the virulence alleles overcoming a major resistance gene to Leptosphaeria maculans in oilseed rape.

Quantitative resistance mediated by multiple genetic factors has been shown to increase the potential for durability of major resistance genes. This was demonstrated in the Leptosphaeria maculans/Brassica napus pathosystem in a 5year recurrent selection field experiment on lines harboring the qualitative resistance gene Rlm6 combined or not with quantitative resistance. The quantitative resistance limited the size of the virulent isolate population. In this study we continued this recurrent selection experiment in the same way to examine whether the pathogen population could adapt and render the major gene ineffective in the longer term. The cultivars Eurol, with a susceptible background, and Darmor, with quantitative resistance, were used. We confirmed that the combination of qualitative and quantitative resistance is an effective approach for controlling the pathogen epidemics over time. This combination did not prevent isolates virulent against the major gene from amplifying in the long term but the quantitative resistance significantly delayed for 5years the loss of effectiveness of the qualitative resistance and disease severity was maintained at a low level on the genotype with both types of resistance after the fungus population had adapted to the major gene. We also showed that diversity of AvrLm6 virulence alleles was comparable in isolates recovered after the recurrent selection on lines carrying either the major gene alone or in combination with quantitative resistance: a single repeat-induced point mutation and deletion events were observed in both situations. Breeding varieties which combine qualitative and quantitative resistance can effectively contribute to disease control by increasing the potential for durability of major resistance genes.

The first meiosis of resynthesized Brassica napus, a genome blender.

Polyploidy promotes the restructuring of merged genomes within initial generations of resynthesized Brassica napus, possibly caused by homoeologous recombination at meiosis. However, little is known about the impact of the first confrontation of two genomes at the first meiosis which could lead to genome exchanges in progeny. Here, we assessed the role of the first meiosis in the genome instability of synthetic B. napus. We used three different newly resynthesized B. napus plants and established meiotic pairing frequencies for the A and C genomes. We genotyped the three corresponding progenies in a cross to a natural B. napus on the two homoeologous A1 and C1 chromosomes. Pairing at meiosis in a set of progenies with various rearrangements was scored. Here, we confirmed that the very first meiosis of resynthesized plants of B. napus acts as a genome blender, with many of the meiotic-driven genetic changes transmitted to the progenies, in proportions that depend significantly on the cytoplasm background inherited from the progenitors. We conclude that the first meiosis generates rearrangements on both genomes and promotes subsequent restructuring in further generations. Our study advances the knowledge on the timing of genetic changes and the mechanisms that may bias their transmission.

Molecular and phenotypic characterization of near isogenic lines at QTL for quantitative resistance to Leptosphaeria maculans in oilseed rape (Brassica napus L.).

The most common and effective way to control phoma stem canker (blackleg) caused by Leptosphaeria maculans in oilseed rape (Brassica napus) is by breeding resistant cultivars. Specific resistance genes have been identified in B. napus and related species but in some B. napus cultivars resistance is polygenic [mediated by quantitative trait loci (QTL)], postulated to be race non-specific and durable. The genetic basis of quantitative resistance in the French winter oilseed rape 'Darmor', which was derived from 'Jet Neuf', was previously examined in two genetic backgrounds. Stable QTL involved in blackleg resistance across year and genetic backgrounds were identified. In this study, near isogenic lines (NILs) were produced in the susceptible background 'Yudal' for four of these QTL using marker-assisted selection. Various strategies were used to develop new molecular markers, which were mapped in these QTL regions. These were used to characterize the length and homozygosity of the 'Darmor-bzh' introgressed segment in the NILs. Individuals from each NIL were evaluated in blackleg disease field trials and assessed for their level of stem canker in comparison to the recurrent line 'Yudal'. The effect of QTL LmA2 was clearly validated and to a lesser extent, QTL LmA9 also showed an effect on the disease level. This work provides valuable material that can be used to study the mode of action of genetic factors involved in L. maculans quantitative resistance.

Detection, introgression and localization of genes conferring specific resistance to Leptosphaeria maculans from Brassica rapa into B. napus.

Blackleg (stem canker) caused by the fungus Leptosphaeria maculans is one of the most damaging diseases of oilseed rape (Brassica napus). Crop relatives represent a valuable source of "new" resistance genes that could be used to diversify cultivar resistance. B. rapa, one of the progenitors of B. napus, is a potential source of new resistance genes. However, most of the accessions are heterozygous so it is impossible to directly detect the plant genes conferring specific resistance due to the complex patterns of avirulence genes in L. maculans isolates. We developed a strategy to simultaneously characterize and introgress resistance genes from B. rapa, by homologous recombination, into B. napus. One B. rapa plant resistant to one L. maculans isolate was used to produce B. rapa backcross progeny and a resynthesized B. napus plant from which a population of doubled haploid lines was derived after crossing with natural B. napus. We then used molecular analyses and resistance tests on these populations to identify and map the resistance genes and to characterize their introgression from B. rapa into B. napus. Three specific genes conferring resistance to L. maculans (Rlm1, Rlm2 and Rlm7) were identified in B. rapa. Comparisons of genetic maps showed that two of these genes were located on the R7 linkage group, in a region homologous to the region on linkage group N7 in B. napus, where these genes have been reported previously. The results of our study offer new perspectives for gene introgression and cloning in Brassicas.

Genetic control of oil content in oilseed rape (Brassica napus L.).

In oilseed rape (Brassica napus L.) like in most oleaginous crops, seed oil content is the main qualitative determinant that confers its economic value to the harvest. Increasing seed oil content is then still an important objective in oilseed rape breeding. In the objective to get better knowledge on the genetic determinism of seed oil content, a genetic study was undertaken in two genetic backgrounds. Two populations of 445 and a 242 doubled haploids (DH) derived from the crosses "Darmor-bzh" x "Yudal" (DY) and "Rapid" x "NSL96/25" (RNSL), respectively, were genotyped and evaluated for oil content in different trials. QTL mapping in the two populations indicate that additive effects are the main factors contributing to variation in oil content. A total of 14 and 10 genomic regions were involved in seed oil content in DY and RNSL populations, respectively, of which five and two were consistently revealed across the three trials performed for each population. Most of the QTL detected were not colocalised to QTL involved in flowering time. Few epistatic QTL involved regions that carry additive QTL in one or the other population. Only one QTL located on linkage group N3 was potentially common to the two populations. The comparisons of the QTL location in this study and in the literature showed that: (i) some of the QTL were more consistently revealed across different genetic backgrounds. The QTL on N3 was revealed in all the studies and the QTL on N1, N8 and N13 were revealed in three studies out of five, (ii) some of the QTL were specific to one genetic background with potentially some original alleles, (iii) some QTL were located in homeologous regions, and (iv) some of the regions carrying QTL for oil content in oilseed rape and in Arabidopsis could be collinear. These results show the possibility to combine favourable alleles at different QTL to increase seed oil content and to use Arabidopsis genomic data to derive markers for oilseed rape QTL and identify candidate genes, as well as the interest to combine information from different segregating populations in order to build a consolidated map of QTL involved in a specific trait.

A new recombined double low restorer line for the Ogu-INRA cms in rapeseed (Brassica napus L.).

A major objective of breeders using the Ogu-INRA cytoplasmic male sterility (cms) system in rapeseed (Brassica napus L.) is to obtain double low restorer lines with a shorter introgression and a good agronomic value. The development of low glucosinolate content (low GC) restorer lines often occurs through the deletion of a part of the introgression. One of these lines has lost the radish Pgi-2 allele expression, without recovering that of the rapeseed Pgi-2 allele. This line shows a defect in the meiotic transmission of the restorer gene Rfo and a very poor agronomic value. We initiated a programme to force non-spontaneous recombination between this Rfo-carrying introgression and the rapeseed homologous chromosome from a low GC B. napus line. Gamma ray irradiation was used to induce chromosome breakage just prior meiosis aiming at just such a recombination. Low GC cms plants were crossed with the pollen of irradiated plants that were heterozygous for this introgression. The F(2) families were scored for their vigour, transmission rate of Rfo and female fertility. One family of plants, R2000, showed an improved behaviour for these three traits. This family presented a unique combination of molecular markers when compared to other rapeseed restorers analysed, which suggests that the recombination event allowed the recovery of B. oleracea genetic information that was originally replaced by the radish introgression in the original restorers. This resulted in a duplicated region (originating from radish and B. oleracea) on the chromosome carrying the introgression in the R2000 family.

A Cluster of Major Specific Resistance Genes to Leptosphaeria maculans in Brassica napus.

ABSTRACT Two types of genetic resistance to Leptosphaeria maculans usually are distinguished in Brassica napus: qualitative, total resistance expressed at the seedling stage and quantitative, partial resistance expressed at the adult plant stage. The latter is under the control of many genetic factors that have been mapped through quantitative trait loci (QTL) studies using 'Darmor' resistance. The former usually is ascribed to race-specific resistance controlled by single resistance to L. maculans (Rlm) genes. Three B. napus-originating specific Rlm genes (Rlm1, Rlm2, and Rlm4) previously were characterized. Here, we report on the genetic identification of two novel resistance genes, Rlm3 and Rlm7, corresponding to the avirulence genes AvrLm3 and AvrLm7. The identification of a novel L. maculans- B. napus specific interaction allowed the detection of another putative new specific resistance gene, Rlm9. The resistance genes were mapped in two genomic regions on LG10 and LG16 linkage groups. A cluster of five resistance genes (Rlm1, Rlm3, Rlm4, Rlm7, and Rlm9) was strongly suggested on LG10. The relation between all these specific resistance genes and their potential role in adult-plant field resistance is discussed. These two Rlm-carrying regions do not correspond to major QTL for Darmor quantitative resistance.

Development of an AFLP-based linkage map and localization of QTLs for seed fatty acid content in condiment mustard (Brassica juncea).

A genetic linkage map of Brassica juncea based on AFLP and RAPD markers was constructed using 131 F1-derived doubled-haploid (DH) plants from a cross between two mustard lines. The map included 273 markers (264 AFLP, 9 RAPD) arranged on 18 linkage groups, and covered a total genetic distance of 1641 cM; 18.3% of the AFLP markers showed a segregation distortion (P < 0.01). The markers with biased segregation were clustered on seven linkage groups. QTLs for oil contents, palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), eicosenoic acid (20:1), and erucic acid (22:1), were mapped on the AFLP linkage map. Correlation studies among fatty acids in the DH population and the localization of QTLs involved in their control indicated that a major gene located on linkage group (LG) 2 controlled the elongation step of erucic acid.

From Arabidopsis thaliana to Brassica napus: development of amplified consensus genetic markers (ACGM) for construction of a gene map.

The evolution of genomes can be studied by comparing maps of homologous genes which show changes in nucleic acid sequences and chromosome rearrangements. In this study, we developed a set of 32 amplified consensus gene markers (ACGMs) that amplified gene sequences from Arabidopsis thaliana and Brassica napus. Our methodology, based on PCR, facilitated the rapid sequencing of homologous genes from various species of the same phylogenetic family and the detection of intragenic polymorphism. We found that such polymorphism principally concerned intron sequences and we used it to attribute a Brassica oleracea or Brassica rapa origin to the B. napus sequences and to map 43 rapeseed genes. We confirm that the genetic position of homologous genes varied between B. napus and A. thaliana. ACGMs are a useful tool for genome evolution studies and for the further development of single nucleotide polymorphism suitable for use in genetic mapping and genetic diversity analyses.

Genetic Control and Host Range of Avirulence Toward Brassica napus Cultivars Quinta and Jet Neuf in Leptosphaeria maculans.

ABSTRACT Leptosphaeria maculans causes blackleg of oilseed rape. Gene-for-gene interactions between race PG3 and Brassica napus cv. Quinta were related to interaction between the fungal avirulence (Avr) gene AvrLm1 and the corresponding resistance gene Rlm1. AvrLm1 isolates were aviru-lent on cvs. Doublol, Vivol, Columbus, and Capitol, and no recombinant phenotypes were observed in the progeny of two AvrLm1 x avrLm1 crosses, suggesting that all of these cultivars may possess Rlm1 or genes displaying the same recognition spectrum, or that a cluster of Avr genes is present at the Avrlm1 locus. In one cross, segregation distortion was observed at the AvrLm1 locus that could be explained by interaction between AvrLm1 and one unlinked deleterious gene, termed Del1. Incompatibility toward cvs. Jet Neuf and Darmor.bzh was governed by a single gene, unlinked to AvrLm1 or Del1. This avirulence gene was termed AvrLm4. Preliminary plant genetic analysis suggested the occurrence of a corresponding dominant resistance gene, termed Rlm4, present in the Quinta line analyzed and linked to Rlm1.

Expression, mapping, and genetic variability of Brassica napus disease resistance gene analogues.

Numerous sequences analogous to resistance (R) genes exist in plant genomes and could be involved in resistance traits. The aim of this study was to identify a large number of Brassica napus sequences related to R genes and also to test the adequacy of specific PCR-based tools for studying them. Different consensus primers were compared for their efficiency in amplifying resistance-gene analogues (RGAs) related to the nucleotide-binding-site subgroup of R genes. Specific primers were subsequently designed to fine-study the different RGAs and we tested their efficiency in three species related to B. napus: Brassica oleracea, Brassica rapa, and Arabidopsis thaliana. Forty-four B. napus RGAs were identified. Among 29 examined, at least one-third were expressed. Eighteen RGAs were mapped on 10 of the 19 B. napus linkage groups. The high variability within these sequences permitted discrimination of each genotype within a B. napus collection. The RGA-specific primers amplified RGAs in the B. oleracea and B. rapa genomes, but the sequences appear to be poorly conserved in A. thaliana. Specific RGA primers are a precise tool for studying known-sequence RGAs. These sequences represent interesting markers that could be correlated with resistance traits in B. napus or related Brassica genomes.

Pathogenicity of Leptosphaeria maculans Isolates on a Brassica napus-B. juncea Recombinant Line.

ABSTRACT The Brassica napus-B. juncea recombinant line (MX), resistant to Leptosphaeria maculans, was produced by interspecific crosses and bears one gene (Jlm1) from the B. juncea B genome. We investigated whether this new resistance was race specific by characterizing protection against a large sample of L. maculans isolates. The pathogenicity of 119 isolates of L. maculans comprising 105 A-group isolates and 14 B-group isolates was studied at the cotyledon stage under controlled conditions using the MX line, the susceptible B. napus cultivar Westar, and the resistant B. juncea cultivar Picra. All but one of the isolates were pathogenic on 'Westar'. Only 3 of the 105 A-group isolates caused very mild symptoms on 'Picra'. Two of these strains were isolated from the MX line and the other from Sinapis arvensis. The other 102 strains caused hypersensitive-type responses. Most B-group isolates were pathogenic on 'Picra'. There were differences in pathogenicity among A-group isolates tested on the MX line, whereas all B-group isolates were pathogenic on this line. A-group isolates obtained from the MX line were more frequently pathogenic on the MX line than those obtained from B. napus cultivars. One isolate from S. arvensis infected the MX line. These results suggest that the resistance of the MX line is unlikely to be durable. Thus, the new resistance gene Jlm1 should probably be used in association with other sources of resistance, in plant breeding schemes, to prevent the breakdown of this resistance.

In vitro androgenesis and segregation distortion inBrassica napus L.: spontaneous versus colchicine-doubled lines.

A total of 750 plantlets were regenerated from 1,400 embryos produced through microspore cultures from one F1 plant of the cross 'Darmor-bzh' x 'Yudal'. Fifty-three percent of the regenerants were evaluated by flow cytometric analysis, which revealed that 31% were spontaneous diploid (SD), 63% were still haploid and the remaining 6% plants had other ploidy levels. Available segregation data (266 markers) produced on this androgenic progeny were used to study the interference between segregation distortion and the mode of chromosome doubling of androgenc lines. On the basis of the present results it is not possible to conclude that distortions are peculiar to one type of regenerated plant; only a difference in the intensity of the bias might be assumed.

Specific molecular marker of the genes controlling linolenic acid content in rapeseed.

In rapeseed, which is an agronomically important oilseed, variation in the linolenic acid content of the oil has been obtained through chemical mutagenesis treatment. Conventional breeding of this quantitative trait, however requires specific molecular markers. By means of biochemical experiments, we have established that the induced variation in linolenic acid content is associated with the fad3 gene encoding the microsomal Δ(15) desaturase. Using a pair of primers specific to this gene and a doubled haploid progeny derived from a low linolenic x high linolenic acid F1hybrid, we have identified a polymorphism of the fad3 alleles between the low- and the high-linolenic acid genotypes. The structure exon/intron of the fad3 DNA sequence seems to be very similar to that of the Arabidopsis fad3 gene. The choice of the primer pair allows specific amplification of one of the two rapeseed fad3 genes. The value and contribution of specific markers to conventional plant breeding is discussed.

Molecular-mapping analysis in Brassica napus using isozyme, RAPD and RFLP markers on a doubled-haploid progeny.

We have undertaken the construction of a Brassica napus genetic map with isozyme (4%), RFLP (26.5%) and RAPD (68%) markers on a 152 lines of a doubled-haploid population. The map covers 1765 cM and comprises 254 markers including three PCR-specific markers and a morphological marker. They are assembled into 19 linkage groups, covering approximatively 71% of the rapeseed genome. Thirty five percent of the studied markers did not segregate according to the expected Mendelian ratio and tended to cluster in eight specific linkage groups. In this paper, the structure of the genetic map is described and the existence of non-Mendelian segregations in linkage analysis as well as the origins of the observed distortions, are discussed. The mapped RFLP loci corresponded to the cDNAs already used to construct B. napus maps. The first results of intraspecific comparative mapping are presented.

Molecular tagging of the dwarf BREIZH (Bzh) gene in Brassica napus.

We mapped the dwarf Bzh gene in B. napus with RAPD and RFLP markers. Research of the linked markers proceeded in two ways: a random approach through the construction of a detailed genetic map and targeting of the dwarf gene using both near-isogenic lines (NILs) and the bulked segregant analysis (BSA) method. The BSA approach was the most efficient in finding DNA markers linked to Bzh, whereas the efficiency of the NILs approach was limited by a too great similarity of the genetic background between the dwarf donor parent and the recurrent lines. Eight RAPD markers were identified as linked to Bzh, the closest being at 0.8±0.7 cM. The random genetic mapping approach added markers and extended the linkage group containing Bzh. This work represents the first step towards a better understanding of the dwarf mutation, the development of marker-assisted selection, and the cloning of the underlying gene responsible for dwarfing.

Identification of RAPD markers linked to a fertility restorer gene for theOgura radish cytoplasmic male sterility of rapeseed (Brassica napus L.).

Bulked segregant analysis was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the restorer gene (Rfo) used in theOgura radish cytoplasmic male sterility of rapeseed. A total of 138 arbitrary 10-mer oligonucleotide primers were screened on the DNA of three pairs of bulks, each bulk corresponding to homozygous restored and male sterile plants of three segregating populations. Six primers produced repeatable polymorphisms between paired bulks. DNA from individual plants of each bulk was then used as a template for amplification with these six primers. DNA polymorphisms generated by four of these primers were found to be completely linked to the restorer gene with the polymorphic DNA fragments being associated either with the fertility restorer allele or with the sterility maintainer allele. Pairwise cross-hybridization demonstrated that the four polymorphic DNA fragments did not share any homology. Southern hybridization of labelled RAPD fragments on digested genomic DNA from the same three pairs of bulks revealed fragments specific to either the male sterile bulks or to the restored bulks and a few fragments common to all bulks, indicating that the amplified sequences are low copy. The four RAPD fragments that were completely linked to the restorer locus have been cloned and sequenced to develop sequence characterized amplified regions (SCARs). This will facilitate the construction of restorer lines used in breeding programs and is the first step towards map-based cloning of the fertility restorer allele.