Development of RAPD markers associated with common bunt resistance to race T1 (Tilletia tritici) in spelt wheat

Common bunt caused by Tilletia tritici and T. laevis has occurred worldwide and reduces yield and quality in common and durum wheats. The development of DNA markers linked to bunt resistance to race T1 in the cross, ‘Laura’(S) בRL5407’ (R), was carried out in this study based on the single head derived F_4:5 and single seed derived F_4:6 populations. Bulked segregant analysis was used to identify two random amplified polymorphic DNA (RAPD) markers linked to the gene for resistance to race T1 in the spelt wheat ‘RL5407′. The two markers identified, UBC548₅₉₀ and UBC274₉₈₈, flanked the resistance gene with a map distance of 9.1 and 18.2 cM, respectively. The former was linked in repulsion phase to bunt resistance while the later was in coupling phase. The two RAPD markers and the common bunt‐resistance gene all segregated in Mendelian fashion. Use of these two RAPD markers together could assist in incorporating the bunt‐resistance gene from spelt wheat into common wheat cultivars by means of marker‐assisted selection.     

User‐friendly markers linked to Fusarium wilt race 1 resistance Fw gene for marker‐assisted selection in pea

Fusarium wilt is one of the most widespread diseases of pea. Resistance to Fusarium wilt race 1 was reported as a single gene, Fw, located on linkage group III. The previously reported AFLP and RAPD markers linked to Fw have limited usage in marker‐assisted selection due to their map distance and linkage phase. Using 80 F₈ recombinant inbred lines (RILs) derived from the cross of Green Arrow × PI 179449, we amplified 72 polymorphic markers between resistant and susceptible lines with the target region amplified polymorphism (TRAP) technique. Marker–trait association analysis revealed a significant association. Five candidate markers were identified and three were converted into user‐friendly dominant SCAR markers. Forty‐eight pea cultivars with known resistant or susceptible phenotypes to Fusarium wilt race 1 verified the marker–trait association. These three markers, Fw_Trap_480, Fw_Trap_340 and Fw_Trap_220, are tightly linked to and only 1.2 cM away from the Fw locus and are therefore ideal for marker‐assisted selection. These newly identified markers are useful to assist in the isolation of the Fusarium wilt race 1 resistance gene in pea.     

A second gene for resistance to race 4 of Fusarium wilt in chickpea and linkage with a RAPD marker

Fusarium wilt caused by Fusarium oxysporum Schlechtend.: Fr f. sp. ciceris (Padwick) Matuo & Sato is a devastating disease of chickpea. The current study was conducted to determine the inheritance of the gene(s) for resistance to race 4 of fusarium wilt and to identify linked RAPD markers using an early wilting line, JG-62, as a susceptible parent. Genetic analysis was performed on the F₁s, F₂s and F₃ families from the cross of JG-62 × Surutato-77. The F₃ families were inoculated with a spore suspension of the race 4 wilt pathogen and the results were used to infer the genotypes of the parent F₂ plants. Results indicated that two independent genes controlled resistance to race 4. Linkage analysis of candidate RAPD marker, CS-27₇₀₀, and the inferred F₂ phenotypic data showed that this marker locus is linked to one of the resistance genes. Allelism indicated that the two resistance sources, Surutato-77 and WR-315, shared common alleles for resistance and the two susceptible genotypes, C-104 and JG-62, carried alleles for susceptibility. The PCR-based marker, CS-27₇₀₀, was previously reported to be linked to the gene for resistance to race 1 in a different population which suggested that the genes for resistance to races 1 and 4 are in close proximity in the Cicer genome. This revised version was published online in August 2006 with corrections to the Cover Date.     

Two genes and linked RAPD markers involved in resistance to Fusarium oxysporum f. sp. Ciceris race 0 in chickpea

The inheritance of resistance to fusarium wilt race 0 of chickpea and linked random amplified polymorphic DNA (RAPD) markers were studied in two F₆:₇ recombinant inbred line (RIL) populations. These RILs were developed from the crosses CA2156 × JG62 (susceptible × resistant) and CA2139 × JG62 (resistant × resistant), and were sown in a field infected with fusarium wilt race 0 in Beja (Tunisia) over 2 years. A1:1 resistant to susceptible ratio was found in the RIL population from the CA2156 × JG62 cross, indicating that a single gene with two alleles controlled resistance. In the second RIL population (CA2139 × JG62) a 3:1 resistant to susceptible ratio indicated that two genes were present and that either gene was sufficient to confer resistance. Linkage analysis showed a RAPD marker, OPJ20600, linked to resistance in both RIL populations, which is present in the resistant parent JG62.     

Inheritance of angular leaf spot resistance in common bean line BAT 332 and identification of RAPD markers linked to the resistance gene

The existence of genetic variability for angular leaf spot (ALS) resistance in the common bean germplasm allows the development of breeding lines resistant to this disease. The BAT 332 line is an important resistance source to common bean ALS. In this work we determined the inheritance pattern and identified RAPD markers linked to a resistance gene present in BAT 332. Populations F₁, F₂,BCs and BCr derived from crosses between BAT 332 and cultivar Rudá were used. Rudá is a commercial cultivar with carioca type grains and susceptible to ALS. The resistance of BAT 332 to race 61.41 of the pathogen was confirmed. Segregation analysis of the plants indicated that a single dominant gene confers resistance. For identification of RAPD markers linked to the resistance gene, bulk segregant analysis (BSA) was used. Two RAPD markers,OPAA07₉₅₀ and OPAO12₉₅₀, linked in coupling phase at 5.10 and 5.83 cM of this gene, respectively, were identified. These molecular markers are important for common bean breeders and geneticists as source of genetic information and for marker assisted selection in breeding programs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Construction of a genetic map of melon with molecular markers and horticultural traits, and localization of genes associated with ZYMV resistance

Abstract: A partial linkage map of melon was constructed from a cross between PI414723 and Dulce. Twenty-two SSR, 46RAPD, 2 ISSR markers and four horticultural markers [female flower form (a), Fusarium resistance, striped epicarp (st), and fruit flesh pH (pH)] were analyzed in an F₂/F₃ population to produce a map spanning 14 linkage groups. We report for the first time map positions for the st, a, and pH genes. One SSR marker was tightly linked to pH. Mapping the a gene for the female flower form to molecular linkage group 4 enabled the merging of the map of horticultural traits with the of molecular markers in this region. Using the 22 SSR markers of this map, two of the three postulated ZYMV resistance genes were located using a BC₁ population (PI414723 recurrent parent). One SSR marker was tightly linked to a ZYMV resistance gene, designated Zym-1. This revised version was published online in August 2006 with corrections to the Cover Date.     

Development and Validation of SCAR Markers Co-Segregating with an Agropyron Elongatum Derived Leaf Rust Resistance Gene Lr24 in Wheat

Summary An Agropyron elongatum-derived leaf rust resistance gene Lr24 located on chromosome 3DL of wheat was tagged with six random amplified polymorphic DNA (RAPD) markers which co-segregated with the gene. The markers were identified in homozygous resistant F₂ plants taken from a population segregating for leaf rust resistance generated from a cross between two near-isogenic lines (NILs) differing only for Lr24. Phenotyping was done by inoculating the plants with pathotype 77-5 of Puccinia triticina. To enable gene-specific selection, three RAPD markers (S1302₆₀₉, S1326₆₁₅ and OPAB-1₃₈₈) were successfully converted to polymorphic sequence characterized amplified region (SCAR) markers, amplifying only the critical DNA fragments co-segregating with Lr24. The SCAR markers were validated for specificity to the gene Lr24 in wheat NILs possessing Lr24 in 10 additional genetic backgrounds including the Thatcher NIL, but not to 43 Thatcher NILs possessing designated leaf rust resistance genes other than Lr24. This indicated the potential usefulness of these SCAR markers in marker assisted selection (MAS) and for pyramiding leaf rust resistance genes in wheat.     

Stability of seed oil quality traits in high and mid-oleic acid sunflower hybrids

Powdery mildew caused by Podosphaera xanthii is an important disease of melon, and race 2F is the predominant race in most areas of China. Resistance to P. xanthii race 2F in melon K7-1 was controlled by a dominant gene, designated Pm-2F, in a 106-member population of recombinant inbred lines derived from K7-1× susceptible K7-2. Using bulked segregant analysis with molecular markers, we have identified two polymorphic simple sequence repeats (SSR) to determine that Pm-2F is located on linkage group II. Comparative genomic analyses using mapped SSR markers and the cucumber genome sequence showed that the melon chromosomal region carrying Pm-2F is homologous to a 288,223 bp genomic region on cucumber chromosome (chr) 1. The SSR markers on chr 1 of cucumber, SSR02734, SSR02733 and CS27 were found linked with Pm-2F. Comparative mapping showed that two SSR markers (SSR02734 and CMBR8) flanked the Pm-2F locus and two nucleotide binding site-leucine-rich repeat resistance genes were identified in the collinear region of cucumber. A cleaved amplified polymorphic sequence (CAPS) marker was developed from the sequence of resistance genes and it delimits the genomic region carrying Pm-2F to 0.8 cM. The evaluation of 165 melon accessions and 13 race differential lines showed that the newly developed CAPS (CAPS-Dde I) marker can be used as a universal marker for effective marker assisted selection in melon powdery mildew resistance breeding. The putative resistance gene cluster provides a potential target site for further fine mapping and cloning of Pm-2F.     

Segregation distortion of Brassica carinata derived black rot resistance in Brassica oleracea

Three segregating F₂ populations were developed by self-pollinating 3 black rot resistant F₁ plants, derived from across between black rot resistant parent line 11B-1-12 and the susceptible cauliflower cultivar ‘Snow Ball’. Plants were wound inoculated using 4 isolates ofXanthomonas campestris pv. campestris (Xcc) race 4, and disease severity ratings of F₂ plants from the three populations were scored. A total of 860 arbitrary oligonucleotide primers were used to amplify DNA from black rot resistant and susceptible F₂ plants and bulks. Eight RAPD markers amplified fragments associated with completely disease free plants following black rot inoculation,which segregated in frequencies far lower than expected. Segregation of markers with black rot resistance indicates that a single, dominant major gene controls black rot resistance in these plants. Stability of this black rot resistance gene in populations derived from 11B-1-12 may complicate introgression into B. oleracea genotypes for hybrid production. This revised version was published online in July 2006 with corrections to the Cover Date.     

Construction of high-resolution linkage map of the Ms locus, a restorer-of-fertility gene in onion (Allium cepa L.)

For the purpose of developing closely-linked molecular markers to the Ms locus, a restorer-of-fertility gene in onions (Allium cepa L.), bulked segregant analysis and randomly amplified polymorphic DNA (RAPD) analyses were utilized. Five RAPD markers polymorphic between male-fertile and male-sterile bulks were identified. These RAPD markers were converted into a simple PCR marker or cleaved amplified polymorphic sequence (CAPS) markers after sequencing the RAPD products and obtaining flanking sequences of the RAPD markers by genome walking. A linkage map was constructed with the Ms locus and flanking markers using a F₂ population. There was no recombinant between the Ms locus and two CAPS markers, jnurf05 and jnurf17. To increase resolution among these closely linked molecular markers and the Ms locus, a total of 1,346 F_2:3 and 2,927 F_2:4 plants were analyzed with two flanking markers for detection of recombinants. Segregation of male-fertility phenotypes in large-sized populations confirmed allelic segregation distortion in favor of the recessive Ms allele. Analysis of the recombinants with closely linked markers revealed only two recombinants between the Ms locus and the jnurf05 markers among 4,273 segregating plants, showing very tight linkage between the two loci. However, linkage disequilibrium between the two loci was not too strong among the breeding lines. Despite weak linkage disequilibrium, these tightly linked markers are useful in accurate marker-assisted selection of the Ms alleles and ultimate isolation of the Ms gene by map-based cloning approach.     

Molecular mapping of Aegilops speltoides derived leaf rust resistance gene Lr28 in wheat

In a segregating homozygous F₂ population of bread wheat involving a leaf rust resistance gene Lr28 derived from Aegilops speltoides, six randomly amplified polymorphic DNA (RAPD) markers, three each in coupling and repulsion phase were identified as linked to Lr28, mapped to a region spanning 32 cM including the locus. The F₂ and F₃ populations were studied in the phytotron challenged with the most virulent pathotype 77-5 of leaf rust. A coupling phase linked RAPD marker S464₇₂₁ and a repulsion phase linked RAPD marker S326₅₅₀ flanked the gene Lr28 by a distance of 2.4± 0.016 cM on either side. The flanking markers genetically worked as co-dominant markers when analyzed together after separate amplification in the F₂ population by distinguishing the homozygotes from the heterozygotes and increased the efficiency of marker assisted selection by reducing the false positives and negatives. One of the three RAPD markers, S421₆₄₀ was converted to locus specific SCAR marker SCS421₆₄₀ which was further truncated by designing primers internal from both ends of the original RAPD amplicon to eliminate a non-specific amplification of nearly same size. The truncated polymorphic sequence characterized amplified region marker (TPSCAR) SCS421₅₇₀ was 70 bp smaller, but resulted in a single band polymorphism specific to Lr28 resistance. The TPSCAR marker was validated for its specificity to the gene Lr28 in nine different genetic backgrounds and on 43 of the 50 Lr genes of both native and alien origin, suggesting the utility of the SCAR markers in pyramiding leaf rust resistance genes in wheat.     

Molecular mapping of black rot resistance locus Xca1bo on chromosome 3 in Indian cauliflower (Brassica oleracea var. botrytis L.)

Black rot is the most devastating disease of cauliflower worldwide causing severe damage to crop. The identification of markers linked to loci that control resistance can facilitate selection of plants for breeding programmes. In the present investigation, F₂ population derived from a cross between ‘Pusa Himjyoti’, a susceptible genotype, and ‘BR‐161’, a resistant genotype, was phenotyped by artificial inoculation using Xcc race 1. Segregation analysis of F₂ progeny indicated that a single dominant locus governed resistance to Xcc race 1 in ‘BR‐161’. Bulk segregant analysis in resistant and susceptible bulks of F₂ progeny revealed seven differentiating polymorphic markers (three RAPD, two ISSR and two SSR) of 102 markers screened. Subsequently, these markers were used to genotype the entire F₂ population, and a genetic linkage map covering 74.7 cM distance was developed. The major locus Xca1bo was mapped in 1.6‐cM interval flanked by the markers RAPD 04₈₃₃ and ISSR 11₆₃₅. The Xca1bo locus was located on chromosome 3. The linked markers will be useful for marker‐assisted resistance breeding in cauliflower.     

Identification and characterization of RAPD and SCAR markers linked to anthracnose resistance gene in sorghum [Sorghum bicolor (L.) Moench]

Anthracnose, one of the destructive foliar diseases of sorghum growing in warm humid regions, is incited by the fungus Colletotrichum graminicola.The inheritance of anthracnose resistance was studied using the parental cultivars of Sorghum bicolor (L.) Moench, HC 136 (susceptible to anthracnose) and G 73 (anthracnose resistant). The F₁ and F₂ plants were inoculated with the local isolates of C. graminicola cultures. The F₂ plants showed a segregation ratio of 3 (susceptible): 1(resistant) indicating that the locus for resistance to anthracnose in sorghum accession G 73 segregates as a recessive trait in a cross to susceptible cultivar HC 136. RAPD (random amplified polymorphic DNA) marker OPJ 01₁₄₃₇ was identified as marker closely linked to anthracnose resistance gene in sorghum by bulked segregant analysis of HC 136 × G73 derived recombinant inbred lines (RILs) of sorghum. A total of 84 random decamer primers were used to screen polymorphism among the parental genotypes. Among these, only 24 primers were polymorphic. On bulked segregant analysis, primer OPJ 01 amplified a 1437 bp fragment only in resistant parent G 73 and resistant bulk. The marker OPJ 01₁₄₃₇ was cloned and sequenced. The sequence of RAPD marker OPJ 01₁₄₃₇ was used to generate specific markers called sequence characterized amplified regions (SCARs). A pair of SCAR markers SCJ 01-1 and SCJ 01-2 was developed using Mac Vector program. SCAR amplification of resistant and susceptible parents along with their respective bulks and RILs confirmed that SCAR marker SCJ 01 is at the same loci as that of RAPD marker OPJ 01₁₄₃₇ and hence, is linked to anthracnose resistance gene. Resistant parent G 73 and resistant bulk amplified single specific band on PCR amplification using SCAR primer pairs. The RAPD marker OPJ 01₁₄₃₇ was mapped at a distance of 3.26 cM apart from the locus governing anthracnose resistance on the sorghum genetic map by the segregation analysis of the RILs. Using BLAST program, it was found that the marker showed 100 per cent alignment with the contig{_}3966 located on the longer arm of chromosome 8 of sorghum genome. Therefore, these identified RAPD and SCAR markers can be used in the resistance-breeding program of sorghum anthracnose by marker-assisted selection.An erratum to this article can be found at     

Analysis of RAPD and AFLP markers linked to resistance to Fusarium oxysporum f. sp. lactucae race 2 in lettuce (Lactuca sativa L.)

Root rot of lettuce, which is caused by Fusarium oxysporum f. sp. lactucae (FOL), is a critical problem in the production of lettuce. FOL-resistant lettuce genetic resources have been identified and used in breeding programs to produce FOL-resistant cultivars. However, the genetic characteristics of resistance genes have not been studied in depth and, therefore, no DNA markers are presently available for these genes. In this study, we analyzed the RRD2 (resistance for root rot disease race 2) locus, which confers resistance to FOL race 2. Resistance loci were analyzed using two cultivars of crisphead lettuce: VP1013 (resistant) and Patriot (susceptible). The segregation patterns of resistant phenotypes in F₂ indicated a single major locus. To define the positions of resistance loci, a linkage map was constructed using amplified fragment length polymorphism and random amplified polymorphic DNA (RAPD) markers. Quantitative trait loci analysis revealed the position of the major resistance locus. A high LOD score was observed for RAPD-marker WF25-42, and this marker showed good correspondence to the phenotype in different cultivars and lines. We successfully developed a sequence characterized amplified region marker from WF25-42.     

Sclerotinia rot resistance in red clover: Identification of RAPD markers using bulked segregant analysis

Random amplified polymorphic DNA (RAPD) was used with the objective of identifying DNA markers linked to the sclerotinia crown and stem rot (SCSR) resistance of red clover. Bulked segregant analysis was used to detect polymorphism that should be linked to SCSR resistance. Two bulks were made by pooling previously extracted DNA. Each bulk (one resistant, and the other susceptible) consisted of eight genotypes from an F₂ population obtained from a cross between a susceptible and a resistant parent. A binomial model was used to select RAPD fragments with a low probability of no linkage with SCSR resistance. Four RAPD fragments were retained as candidate markers of SCSR resistance. Three are associated with resistance and one with susceptibility.     

Identification of PCR-based markers linked to the powdery-mildew-resistance gene Pl1 from Malus robusta in cultivated apple

The availability of molecular markers linked to mildew resistance genes would enhance the efficiency of apple-breeding programmes. This investigation focuses on the identification of random amplified polymorphic DNA (RAPD) markers linked to the Pl₁ gene for mildew resistance, which has introgressed from Malus robusta into cultivated apples. The RAPD marker technique was combined with a modified ‘bulked seg-regant analysis’ mapping strategy. About 850 random decamer primers used as single primers or in combinations were tested by PCR analysis on the basis of resistant and susceptible DNA pools. Selected primers producing RAPD fragments were applied in an additional selection step to M. robusta and genotypes representing intermediate breeding stages of the breeding population 93/9, for which a 1:1 segregation could be observed for the resistance trait. Seven RAPD markers, all representing introgressed DNA sequences from M. robusta, were identified and arranged with the Pl₁ locus in a common linkage group. The two most tightly-linked RAPD markers, OPAT20₄₅₀ and OPD2₁₀₀₀ were mapped with a genetic distance of 4.5 and 5 cM, respectively, from the Pl₁ gene. Both markers are suitable for marker-assisted selection in apple breeding. The polymorphic DNA fragment OPAT20₄₅₀ was cloned and sequenced, and longer primers for the generation of a sequence-characterized amplified region (SCAR) marker have been constructed; this marker was easier to score than the original RAPD marker.     

Identification of a molecular marker linked to an Agropyron elongatum-derived gene Lr19 for leaf rust resistance in wheat

The leaf rust resistance gene Lr19, transferred from Agropyron elongatum into wheat (Triticum aestivum L.) imparts resistance to all pathotypes of leaf rust (Puccinia recondita f.sp. tritici) in South‐east Asia. A segregating F₂ population from a cross between the leaf rust resistant parent ‘HW 2046’ carrying Lr19 and a susceptible parent ‘Agra Local’ was screened in the phytotron against a virulent pathotype 77‐5 of leaf rust with the objective of identifying the molecular markers linked to Lr19. The gene was first tagged with a randomly amplified polymorphic DNA (RAPD) marker S73₇₂₈. The RAPD marker linked to the gene Lr19 which mapped at 6.4 ± 0.035 cM distance, was converted to a sequence characterized amplified region (SCAR) marker. The SCAR marker (SCS73₇₁₉) was specific to Lr19 and was not amplified in the near‐isogenic lines (NILs) carrying other equally effective alien genes Lr9, Lr28 and Lr32 enabling breeders to pyramid Lr19 with these genes.     

Molecular markers for yellow stem borer Scirpophaga incertulas (Walker) resistance in rice

Breeding for yellow stem borer resistance in rice is difficult owing to the complex genetics of the trait and inherent difficulties in screening. Identification of molecular markers linked to the trait would enhance phenotypic evaluation for the trait. An F₂ population was developed using parents contrasting in their reaction to yellow stem borer resistance. Random Amplified Polymorphic DNA (RAPD) analysis,in conjunction with bulked segregant analysis, enabled us to identify four phenotype-specific RAPD markers. The markers C1₃₂₀ and K6₆₉₅ were linked with resistance and AH5₆₆₀ and C4₁₃₀₀ with susceptibility. The markers K6₆₉₅ and AH5₆₆₀ were linked to the gene(s) at distances of 12.8 cM and 14.9 cM, respectively. Scoring of these markers in a set of germplasm confirmed their reproducibility and their association with the trait. This revised version was published online in August 2006 with corrections to the Cover Date.     

Development of a SCAR marker linked with a MYMV resistance gene in mungbean (Vigna radiata L. Wilczek)

Yellow mosaic disease (YMD) caused by mungbean yellow mosaic virus (MYMV) is the most important disease of mungbean, causing great yield loss. The present investigation was carried out to study the inheritance and identify molecular markers linked with MYMV resistance gene by using F₁, F₂ and 167 F_2 : 8 recombinant inbred lines (RILs) developed from the cross ‘TM‐99‐37’ (resistant) × Mulmarada (susceptible). The F₁ was susceptible, F₂ segregated in 3S:1R phenotypic ratio and RILs segregated in 1S:1R ratio in the field screening indicating that the MYMV resistance gene is governed by a single recessive gene. Of the 140 RAPD primers, 45 primers showing polymorphism in parents were screened using bulked segregant analysis. Three primers amplified specific polymorphic fragments viz. OPB‐07_600, OPC‐06₁₇₅₀ and OPB‐12₈₂₀. The marker OPB‐07₆₀₀ was more closely linked (6.8 cM) with a MYMV resistance gene as compared to OPC‐06₁₇₅₀ (22.8 cM) and OPB‐12₈₂₀ (25.2 cM). The resistance‐specific fragment OPB‐07₆₀₀ was cloned, sequenced and converted into a sequence‐characterized amplified region (SCAR) marker and validated in twenty genotypes with different genetic backgrounds.     

Identification of RAPD markers linked to the rust (<Emphasis Type="Italic">Uromyces fabae</Emphasis>) resistance gene in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>)

Summary Two RAPD markers linked to gene for resistance (assayed as pustule number cm⁻² leaf area) to rust [Uromyces fabae (Pers.) de Bary] in pea (Pisum sativum L.) were identified using a mapping population of 31 BC₁F₁ [HUVP 1 (HUVP 1 × FC 1] plants, FC 1 being the resistant parent. The analysis of genetics of rust resistance was based on the parents, F₁, F₂, BC₁F₁ and BC₁F₂ generations. Rust resistance in pea is of non-hypersensitive type; it appeared to be governed by a single partially dominant gene for which symbol Ruf is proposed. Further, this trait seems to be affected by some polygenes in addition to the proposed oligogene Ruf. A total of 614 decamer primers were used to survey the parental polymorphism with regard to DNA amplification by polymerase chain reaction. The primers that amplified polymorphic bands present in the resistant parent (FC 1) were used for bulked segregant analysis. Those markers that amplified consistently and differentially in the resistant and susceptible bulks were separately tested with the 31 BC₁F₁ individuals. Two RAPD makers, viz., SC10-82₃₆₀ (primer, GCCGTGAAGT), and SCRI-71₁₀₀₀ (primer, GTGGCGTAGT), flanking the rust resistance gene (Ruf) with a distance of 10.8 cM (0.097 rF and LOD of 5.05) and 24.5 cM (0.194 rF and a LOD of 2.72), respectively, were identified. These RAPD markers were not close enough to Ruf to allow a dependable maker-assisted selection for rust resistance. However, if the two makers flanking Ruf were used together, the effectiveness of MAS would be improved considerably.