Development of SCAR markers linked to the gene Or5 conferring resistance to broomrape (Orobanche cumana Wallr.) in sunflower

A consensus molecular linkage map of 61.9 cM containing the Or5 gene, which confers resistance to race E of broomrape orobanche cumana, five SCAR markers (three dominant, two codominant) and one RAPD marker were identified based on segregation data scored from two F₂ populations of susceptible×resistant sunflower line crosses. Bulked segregant analysis was carried out to generate the five SCAR markers, while the single RAPD marker in the group was identified from 61 segregating RAPD markers that were directly screened on one of the two F₂ populations. The five SCAR markers, RTS05, RTS28, RTS40, RTS29 and RTS41, were significantly (LOD≥4.0) linked to the Or5 gene and mapped separately at 5.6, 13.6, 14.1, 21.4 and 39.4 cM from the Or5 locus on one side, while the RAPD marker, UBC120_660, was found at 22.5 cM (LOD=1.4) on the opposite side. These markers should facilitate the efficient transfer of the resistance gene among sunflower breeding lines. As the first report on molecular markers linked to a broomrape resistance gene, the present work provides a starting point to study other genes and to examine the hypothesis of the clustering of broomrape resistance genes in sunflower. Received: 16 September 1998 / Accepted: 22 June 1999     

Molecular mapping of the rust resistance gene <Emphasis Type="Italic">R</Emphasis><Subscript><Emphasis Type="Italic">4</Emphasis></Subscript> to a large NBS-LRR cluster on linkage group 13 of sunflower

Rust is a serious fungal disease in the sunflower growing areas worldwide with increasing importance in North America in recent years. Several genes conferring resistance to rust have been identified in sunflower, but few of them have been genetically mapped and linked to molecular markers. The rust resistance gene R ₄ in the germplasm line HA-R3 was derived from an Argentinean open-pollinated variety and is still one of most effective genes. The objectives of this study were to determine the chromosome location of the R ₄ gene and the allelic relationship of R ₄ with the R _adv rust resistance gene. A total of 63 DNA markers previously mapped to linkage group (LG) 13 were used to screen for polymorphisms between two parental lines HA 89 and HA-R3. A genetic map of LG 13 was constructed with 21 markers, resulting in a total map length of 93.8 cM and an average distance of 4.5 cM between markers. Two markers, ZVG61 and ORS581, flanked the R ₄ gene at 2.1 and 0.8 cM, respectively, and were located on the lower end of LG 13 within a large NBS-LRR cluster identified previously. The PCR pattern generated by primer pair ZVG61 was unique in the HA-R3 line, compared to lines HA-R1, HA-R4, and HA-R5, which carry other R ₄ alleles. A SCAR marker linked to the rust resistance gene R _adv mapped to LG 13 at 13.9 cM from the R ₄ locus, indicating that R _adv is not an allele of the R ₄ locus. The markers tightly linked to the R ₄ gene will facilitate gene pyramiding for rust resistance breeding of sunflower.     

Development of RAPD and SCAR markers linked to the Russian wheat aphid resistance gene Dn2 in wheat

 RAPD (random amplified polymorphic DNA) analysis was used to identify molecular markers linked to the Dn2 gene conferring resistance to the Russian wheat aphid (Diuraphis noxia Mordvilko). A set of near-isogenic lines (NILs) was screened with 300 RAPD primers for polymorphisms linked to the Dn2 gene. A total of 2700 RAPD loci were screened for linkage to the resistance locus. Four polymorphic RAPD fragments, two in coupling phase and two in repulsion phase, were identified as putative RAPD markers for the Dn2 gene. Segregation analysis of these markers in an F₂ population segregating for the resistance gene revealed that all four markers were closely linked to the Dn2 locus. Linkage distances ranged from 3.3 cM to 4.4 cM. Southern analysis of the RAPD products using the cloned RAPD markers as probes confirmed the homology of the RAPD amplification products. The coupling-phase marker OPB10_880c and the repulsion-phase marker OPN1_400r were converted to sequence characterized amplified region (SCAR) markers. SCAR analysis of the F₂ population and other resistant and susceptible South African wheat cultivars corroborated the observed linkage of the RAPD markers to the Dn2 resistance locus. These markers will be useful for marker-assisted selection of the Dn2 gene for resistance breeding and gene pyramiding. Received: 1 July 1997 / Accepted: 20 October 1997     

Molecular tagging of a novel rust resistance gene R 12 in sunflower (Helianthus annuus L.)

Sunflower production in North America has recently suffered economic losses in yield and seed quality from sunflower rust (Puccinia helianthi Schwein.) because of the increasing incidence and lack of resistance to new rust races. RHA 464, a newly released sunflower male fertility restorer line, is resistant to both of the most predominant and most virulent rust races identified in the Northern Great Plains of the USA. The gene conditioning rust resistance in RHA 464 originated from wild Helianthus annuus L., but has not been molecularly marked or determined to be independent from other rust loci. The objectives of this study are to identify molecular markers linked to the rust resistance gene and to investigate the allelism of this gene with the unmapped rust resistance genes present in HA-R6, HA-R8 and RHA 397. Virulence phenotypes of seedlings for the F₂ population and F_2:3 families suggested that a single dominant gene confers rust resistance in RHA 464, and this gene was designated as R ₁₂ . Bulked segregant analysis identified ten markers polymorphic between resistant and susceptible bulks. In subsequent genetic mapping, the ten markers covered 33.4 cM of genetic distance on linkage group 11 of sunflower. A co-dominant marker CRT275-11 is the closest marker distal to R ₁₂ with a genetic distance of 1.0 cM, while ZVG53, a dominant marker linked in the repulsion phase, is proximal to R ₁₂ with a genetic distance of 9.6 cM. The allelism test demonstrated that R ₁₂ is not allelic to the rust resistance genes in HA-R6, HA-R8 and RHA 397, and it is also not linked to any previously mapped rust resistance genes. Discovery of the R ₁₂ novel rust resistance locus in sunflower and associated markers will potentially support the molecular marker-assisted introgression and pyramiding of R ₁₂ into sunflower breeding lines.     

Characterization and fine mapping of <Emphasis Type="Italic">RppQ</Emphasis>, a resistance gene to southern corn rust in maize

Southern corn rust (SCR) is a fungal disease caused by Puccinia polysora Underw, which can infect maize and may result in substantial yield losses in maize production. The maize inbred line Qi319 carries the SCR resistance gene RppQ. In order to identify molecular markers linked to the RppQ gene, several techniques were utilized including random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), and amplified fragment length polymorphism (AFLP). In addition, sequence characterized amplified region (SCAR) techniques combined with bulked segregant analysis (BSA) were used. Seven RAPD markers, eight SSR markers, and sixty-three AFLP primer combinations amplified polymorphisms between two parents and two bulk populations. A large F₂ population was used for genetic analysis and for fine mapping of the RppQ gene region. One AFLP polymorphic band, M-CAA/E-AGC₃₂₄, was converted to a SCAR marker, MA7, which was mapped to a position 0.46 cM from RppQ. Finally, the RppQ gene was mapped between the SCAR marker MA7 and the AFLP marker M-CCG/E-AGA₁₅₇ with distances of 0.46 and 1.71 cM, respectively.     

Identification and validation of molecular markers linked to the leaf rust resistance gene Lr19 in wheat

A leaf rust resistance gene Lr19 on the chromosome 7DL of wheat derived from Agropyron elongatum was tagged with random amplified polymorphic DNA (RAPD) and microsatellite markers. The F₂ population of 340 plants derived from a cross between the leaf rust resistant near-isogenic line (NIL) of Thatcher (Tc + Lr19) and leaf rust susceptible line Agra Local that segregated for dominant monogenic leaf rust resistance was utilized for generating the mapping population. The molecular markers were mapped in the F₂ derived F₃ homozygous population of 140 seedlings. Sixteen RAPD markers were identified as linked to the alien gene Lr19 among which eight were in a coupling phase linkage. Twelve RAPD markers co-segregated with Lr19 locus. Nine microsatellite markers located on the long arm of chromosome 7D were also mapped as linked to the gene Lr19, including 7 markers which co-segregated with Lr19 locus, thus generating a saturated region carrying 25 molecular markers linked to the gene Lr19 within 10.2 ± 0.062 cM on either side of the locus. Two RAPD markers S265₅₁₂ and S253₇₃₇ which flanked the locus Lr19 were converted to sequence characterized amplified region markers SCS265₅₁₂ and SCS253₇₃₆, respectively. The marker SCS265₅₁₂ was linked with Lr19 in a coupling phase and the marker SCS253₇₃₆ was linked in a repulsion phase, which when used together mimicked one co-dominant marker capable of distinguishing the heterozygous resistant seedlings from the homozygous resistant. The molecular markers were validated on NILs mostly in Thatcher background isogenic for 44 different Lr genes belonging to both native and alien origin. The validation for polymorphism in common leaf rust susceptible cultivars also confirmed the utility of these tightly linked markers to the gene Lr19 in marker-assisted selection.     

RFLP and RAPD mapping of the sunflower Pl1 locus for resistance to Plasmopara halstedii race 1

The Pl1 locus in sunflower, Helianthus annuus L., conferring resistance to downy mildew, Plasmopara halstedii, race 1 has been located in linkage group 1 of the consensus RFLP map of the cultivated sunflower. Bulked segregant analyses were used on 135 plants of an F₂ progeny from a cross between a downy mildew susceptible line, GH, and RHA266, a line carrying Pl1. Two RFLP markers and one RAPD marker linked to the Pl1 locus have been identified. The RFLP markers are located at 5.6 cM and 7.1 cM on either side of Pl1. The RAPD marker is situated at 43.7 cM from Pl1. The significance and applications of these markers in sunflower breeding are discussed.     

Development of SRAP, SRAP-RGA, RAPD and SCAR markers linked with a Fusarium wilt resistance gene in eggplant

Fusarium wilt (Fusarium oxysporum Schlecht. f. sp. melongenae) is a vascular disease of eggplant (Solanum melongena L.). The objectives of this work were (1) to confirm the monogenic inheritance of fusarium wilt resistance in eggplant, (2) to identify molecular markers linked to this resistance, and (3) to develop SCAR markers from most informative markers. We report the tagging of the gene for resistance to fusarium wilt (FOM) in eggplant using SRAP, RGA, SRAP-RGA and RAPD markers. Analysis of segregation data confirmed the monogenic inheritance of resistance. DNA from F₂ and BC₁ populations of eggplant segregating for fusarium wilt resistance was screened with 2,316 primer combinations to detect polymorphism. Three markers were linked within 2.6 cM of the gene. The codominant SRAP marker Me8/Em5 and dominant SRAP-RGA marker Em12/GLPL2 were tightly linked to each other and mapped 1.2 cM from the resistance gene, whereas RAPD marker H12 mapped 2.6 cM from the gene and on the same side as the other two markers. The SRAP marker was converted into two dominant SCAR markers that were confirmed to be linked to the resistance gene in the F_2, BC₁ and F₂ of BC₃ generations of the same cross. These markers provide a starting point for mapping the eggplant FOM resistance gene in eggplant and for exploring the synteny between solanaceous crops for fusarium wilt resistance genes. The SCAR markers will be useful for identifying fusarium wilt-resistant genotypes in marker-assisted selection breeding programs using segregating progenies of the resistant eggplant progenitor used in this study.     

Molecular mapping of a rust resistance gene <Emphasis Type="Italic">R</Emphasis><Subscript><Emphasis Type="Italic">14</Emphasis></Subscript> in cultivated sunflower line PH 3

Sunflower, the fifth largest oilseed crop in the world, plays an important role in human diets. Recently, sunflower production in North America has suffered serious yield losses from newly evolved races of sunflower rust (Puccinia helianthi Schwein.). The rust resistance gene, designated R ₁₄ , in a germplasm line PH 3 originated from a wild Helianthus annuus L. population resistant to 11 rust races. PH 3 has seedling with an extraordinary purple hypocotyl color. The objectives of this study were to map both the R ₁₄ rust resistance gene and the purple hypocotyl gene-designated PHC in PH 3, and to identify molecular markers for marker-assisted breeding for sunflower rust resistance. A set of 517 mapped SSR/InDel and four SNP markers was used to detect polymorphisms between the parents. Fourteen markers covering a genetic distance of 17.0 cM on linkage group (LG) 11 were linked to R ₁₄ . R ₁₄ was mapped to the middle of the LG, with a dominant SNP marker NSA_000064 as the closest marker at a distance of 0.7 cM, and another codominant marker ORS542 linked at 3.5 cM proximally. One dominant marker ZVG53 was linked on the distal side at 6.9 cM. The PHC gene was also linked to R ₁₄ with a distance of 6.2 cM. Chi-squared analysis of the segregation ratios of R ₁₄ , PHC, and ten linked markers indicated a deviation from an expected 1:2:1 or 3:1 ratio. The closely linked molecular or morphological markers could facilitate sunflower rust-resistant breeding and accelerate the development of rust-resistant hybrids.     

Identification and validation of RAPD and SCAR markers linked to the gene <Emphasis Type="Italic">Er3</Emphasis> conferring resistance to <Emphasis Type="Italic">Erysiphe pisi</Emphasis> DC in pea

Three genes, er1, er2 and Er3, conferring resistance to powdery mildew (Erysiphe pisi) in pea have been described so far. Because single gene-controlled resistance tends to be overcome by evolution of pathogen virulence, accumulation of several resistance genes into a single cultivar should enhance the durability of the resistance. Molecular markers linked to genes controlling resistance to E. pisi may facilitate gene pyramiding in pea breeding programs. Molecular markers linked to er1 and er2 are available. In the present study, molecular markers linked to Er3 have been obtained. A segregating F₂ population derived from the cross between a breeding line carrying the Er3 gene, and the susceptible cultivar ‘Messire’ was developed and genotyped. Bulk Segregant Analysis (BSA) was used to identify Random Amplified Polymorphic DNA (RAPD) markers linked to Er3. Four RAPD markers linked in coupling phase (OPW04_637, OPC04_640, OPF14_1103, and OPAH06_539) and two in repulsion phase (OPAB01_874 and OPAG05_1240), were identified. Two of these, flanking Er3, were converted to Sequence Characterized Amplified Region (SCAR) markers. The SCAR marker SCW4₆₃₇ co-segregated with the resistant gene, allowing the detection of all the resistant individuals. The SCAR marker SCAB1₈₇₄, in repulsion phase with Er3, was located at 2.8 cM from the gene and, in combination with SCW4₆₃₇, was capable to distinguish homozygous resistant individuals from heterozygous with a high efficiency. In addition, the validation for polymorphism in different genetic backgrounds and advanced breeding material confirmed the utility of both markers in marker-assisted selection.     

Identification of molecular markers for the detection of the yellow rust resistance gene Yr17 in wheat

The Yr17 gene, which is present in many European wheat cultivars, displays yellow rust resistance at the seedling stage. The gene introduced into chromosome 2A from Aegilops ventricosa was previously found to be closely linked (0.5 cM) to leaf and stem rust resistance genes Lr37 and Sr38, respectively. The objective of this study was to identify molecular markers linked to the Yr17 gene. We screened with RAPD primers, for polymorphism, the DNAs of cv. Thatcher and the leaf rust-resistant near-isogenic line (NIL) RL 6081 of cv. Thatcher carrying the Lr37 gene. Using a F2 progeny of the cross between VPM1 (resistant) and Thésée (susceptible), the RAPD marker OP-Y15580 was found to be closely linked to the Yr17 gene. We converted the OP- Y15580 RAPD marker into a sequence characterized amplified region (SCAR). This SCAR marker (SC-Y15) was linked at 0.8 ± 0.7 cM to the Yr17 resistance gene. We tested the SC-Y15 marker over a survey of 37 wheat cultivars in order to verify its consistency in different genetic backgrounds and to explain the resistance of some cultivars against yellow rust. Moreover, we showed that the Xpsr150-2Mv locus marker of Lr gene described by Bonhomme et al. [6] which possesses A. ventricosa introgression on the 2A chromosome was also closely linked to the Yr17 gene. Both the SCAR SC-Y15 and Xpsr150-2Mv markers should be used in breeding programmes in order to detect the cluster of the three genes Yr17, Lr37 and Sr38 in cross progenies. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular markers for rust and pyricularia leaf spot disease resistance in pearl millet

 Pearl millet [Pennisetum glaucum (L.) R.Br.] is a warm-season grass used for food, feed, fodder and forage, primarily in countries of Africa and India but grown around the world. The two most-destructive diseases to pearl millet in the United States are rust (caused by Puccinia substriata var. indica) and pyricularia leaf spot (caused by Pyricularia grisea). Genes for disease resistance to both pathogens have been transferred into agronomically acceptable forage and grain cultivars. A study was undertaken to identify molecular markers for three rust loci and one pyricularia resistance locus. Three segregating populations were screened for RAPDs using random decamer primers and for RFLPs using a core set of probes detecting single-copy markers on the pearl millet map. The rust resistance gene Rr ₁ from the pearl millet subspecies P. glaucum ssp. monodii was linked 8.5 cM from the RAPD OP-G8₃₅₀. The linkage of two RFLP markers, Xpsm108 (15.5 cM) and Xpsm174 (17.7 cM), placed the Rr ₁ gene on linkage-group 3 of the pearl millet map. Rust resistance genes from both Tift 89D₂ and ICMP 83506 were placed on linkage-group 4 by determining genetic linkage to the RFLP marker Xpsm716 (4.9 and 0.0 cM, respectively). Resistance in ICMP 83506 was also linked to the RFLP marker Xpsm306 (10.0 cM), while resistance in Tift 89D₂ was linked to RAPD markers OP-K19₃₅₀ (8.8 cM) and OP-O8₃₅₀ (19.6 cM). Fragments from OP-K19 and OP-O8 in the ICMP 83506 population, and Xpsm306 in the Tift 89D₂ population, were monomorphic. Only one RAPD marker (OP-D11₇₀₀, 5.6 cM) was linked to pyricularia leaf spot resistance. Attempts to detect polymorphisms with rice RFLP probes linked to rice blast resistance (Pyricularia oryzae; syn=P. grisea) were unsuccessful. Received: 19 May 1997 / Accepted: 21 October 1997     

Downy mildew (Pl ( 8 ) and Pl ( 14 )) and rust (R ( Adv )) resistance genes reside in close proximity to tandemly duplicated clusters of non-TIR-like NBS-LRR-encoding genes on sunflower chromosomes 1 and 13

Nucleotide binding site-leucine rich repeat (NBS-LRR) proteins are encoded by a ubiquitous gene family in sunflower and frequently harbor disease resistance genes. We investigated NBS-LRR-encoding resistance gene candidates (RGCs) flanking the downy mildew resistance genes Pl ₈ and Pl ₁₄ and the rust resistance gene R _Adv , which map on the NBS-LRR clusters of linkage groups 1 and 13 in sunflower genome. We shotgun sequenced bacterial artificial chromosome (BAC) clones proximal to Pl ₈ , Pl ₁₄ , and R _Adv and identified seven novel non-Toll/interleukin-1 receptor (TIR)-like NBS-LRR RGCs, which clustered with previously identified RGCs of linkage group 13 but were phylogenetically distant from the TIR- and non-TIR-NBS-LRR-encoding superfamilies of sunflower. Six of the seven predicted RGCs have intact open reading frames and reside in genomic segments with abundant transposable elements. The genomic localization and sequence similarity of the novel non-TIR-like predicted RGCs suggests that they originated from tandem duplications. RGCs in the proximity of Pl ₈ and R _Adv were likely introgressed from silverleaf sunflower genome, where the RGC cluster of linkage group 13 is duplicated in two independent chromosomes that have different architecture and level of recombination from the respective common sunflower chromosomes.     

Development of SCAR markers linked to male sterility and very high linoleic acid content in safflower

Practically no molecular tools have been developed so far for safflower (Carthamus tinctorius L.) breeding. The objective of the present research was to develop molecular markers for the closely linked genes Li, controlling very high linoleic acid content, and Ms, controlling nuclear male sterility (NMS). A mapping population of 162 individuals was developed from the NMS line CL1 (64–79% linoleic acid) and the line CR-142 (84–90%), and phenotyped in the F₂ and F₃ generations. Bulked segregant analysis with random amplified polymorphic (RAPD) markers revealed linkage of five RAPD bands to the Li and Ms genes. RAPD fragments were converted into sequence-characterized amplified region (SCAR) markers. A linkage map including the five SCAR markers and the Li and Ms genes was constructed. SCAR markers flanked both loci at minimum distances of 15.7 cM from the Li locus and 3.7 cM from the Ms locus. These are the first molecular markers developed for trait selection in safflower.     

RAPD-based SCAR marker SCA 12 linked to recessive gene conferring resistance to anthracnose in sorghum [Sorghum bicolor (L.) Moench]

Anthracnose, caused by Colletotrichum graminicola, infects all aerial parts of sorghum, Sorghum bicolor (L.) Moench, plants and causes loss of as much as 70%. F₁ and F₂ plants inoculated with local isolates of C. graminicola indicated that resistance to anthracnose in sorghum accession G 73 segregated as a recessive trait in a cross with susceptible cultivar HC 136. To facilitate the use of marker-assisted selection in sorghum breeding programs, a PCR-based specific sequence characterized amplified region (SCAR) marker was developed. A total of 29 resistant and 20 susceptible recombinant inbred lines (RILs) derived from a HC 136 × G 73 cross was used for bulked segregant analysis to identify a RAPD marker closely linked to a gene for resistance to anthracnose. The polymorphism between the parents HC 136 and G 73 was evaluated using 84 random sequence decamer primers. Among these, only 24 primers generated polymorphism. On bulked segregant analysis, primer OPA 12 amplified a unique band of 383 bp only in the resistant parent G 73 and resistant bulk. Segregation analysis of individual RILs showed the marker OPA 12₃₈₃ was 6.03 cM from the locus governing resistance to anthracnose. The marker OPA 12₃₈₃ was cloned and sequenced. Based on the sequence of cloned RAPD product, a pair of SCAR markers SCA 12-1 and SCA 12-2 was designed using the MacVector program, which specifically amplified this RAPD fragment in resistant parent G 73, resistant bulk and respective RILs. Therefore, it was confirmed that SCAR marker SCA 12 is at the same locus as RAPD marker OPA 12₃₈₃ and hence, is linked to the gene for resistance to anthracnose.     

Molecular mapping of <Emphasis Type="Italic">Stb1</Emphasis>, a potentially durable gene for resistance to septoria tritici blotch in wheat

Septoria tritici blotch (STB), caused by the ascomycete Mycosphaerella graminicola (anamorph Septoria tritici), was the most destructive disease of wheat in Indiana and adjacent states before deployment of the resistance gene Stb1 during the early 1970s. Since then, Stb1 has provided durable protection against STB in widely grown wheat cultivars. However, its chromosomal location and allelic relationships to most other STB genes are not known, so the molecular mapping of Stb1 is of great interest. Genetic analyses and molecular mapping were performed for two mapping populations. A total of 148 F₁ plants (mapping population I) were derived from a three-way cross between the resistant line P881072-75-1 and the susceptible lines P881072-75-2 and Monon, and 106 F₆ recombinant-inbred lines (mapping population II) were developed from a cross between the resistant line 72626E2-12-9-1 and the susceptible cultivar Arthur. Bulked-segregant analysis with random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and microsatellite or simple-sequence repeat (SSR) markers was conducted to identify those that were putatively linked to the Stb1 gene. Segregation analyses confirmed that a single dominant gene controls the resistance to M. graminicola in each mapping population. Two RAPD markers, G7₁₂₀₀ and H19₅₂₀, were tightly linked to Stb1 in wheat line P881072-75-1 at distances of less than 0.68 cM and 1.4 cM, respectively. In mapping population II, the most closely linked marker was SSR Xbarc74, which was 2.8 cM proximal to Stb1 on chromosome 5BL. Microsatellite loci Xgwm335 and Xgwm213 also were proximal to Stb1 at distances of 7.4 cM and 8.3 cM, respectively. The flanking AFLP marker, EcoRI-AGC/MseI-CTA-1, was 8.4 cM distal to Stb1. The two RAPD markers, G7₁₂₀₀ and H19₅₂₀, and AFLP EcoRI-AGC/MseI-CTA-1, were cloned and sequenced for conversion into sequence-characterized amplified region (SCAR) markers. Only RAPD allele H19₅₂₀ could be converted successfully, and none of the SCAR markers was diagnostic for the Stb1 locus. Analysis of SSR and the original RAPD primers on several 5BL deletion stocks positioned the Stb1 locus in the region delineated by chromosome breakpoints at fraction lengths 0.59 and 0.75. The molecular markers tightly linked to Stb1 could be useful for marker-assisted selection and for pyramiding of Stb1 with other genes for resistance to M. graminicola in wheat.     

Identification and mapping on chromosome 9 of RAPD markers linked to Sw-5 in tomato by bulked segregant analysis

Bulked segregant analysis was used to identify random amplified polymorphic DNA (RAPD) markers linked to the Sw-5 gene for resistance to tomato spotted wilt virus (TSWV) in tomato. Using two pools of phenotyped individuals from one segregating population, we identified four RAPD markers linked to the gene of interest. Two of these appeared tightly linked to Sw-5, whereas another, linked in repulsion phase, enabled the identification of heterozygous and susceptible plants. After linkage analysis of an F₂ population, the RAPD markers were shown to be linked to Sw-5 within a distance of 10.5 cM. One of the RAPD markers close to Sw-5 was used to develop a SCAR (sequence characterized amplified region) marker. Another RAPD marker was stabilized into a pseudo-SCAR marker by enhancing the specificity of its primer sequence without cloning and sequencing. RAPD markers were mapped to chromosome 9 on the RFLP tomato map developed by Tanksley et al. (1992). The analysis of 13 F₃ families and eight BC₂ populations segregating for resistance to TSWV confirmed the linkage of the RAPD markers found. These markers are presently being used in marker-assisted plant breeding.     

Genetic mapping of rust resistance genes in confection sunflower line HA-R6 and oilseed line RHA 397

Few widely effective resistance sources to sunflower rust, incited by Puccinia helianthi Schwein., have been identified in confection sunflower (Helianthus annuus L.). The USDA inbred line HA-R6 is one of the few confection sunflower lines resistant to rust. A previous allelism test indicated that rust resistance genes in HA-R6 and RHA 397, an oilseed-type restorer line, are either allelic or closely linked; however, neither have been characterized nor molecularly mapped. The objectives of this study are (1) to locate the rust resistance genes in HA-R6 and RHA 397 on a molecular map, (2) to develop closely linked molecular markers for rust resistance diagnostics, and (3) to determine the resistance spectrum of two lines when compared with other rust-resistant lines. Two populations of 140 F_2:3 families each from the crosses of HA 89, as susceptible parent, with HA-R6 and RHA 397 were inoculated with race 336 of P. helianthi in the greenhouse. The resistance genes (R-genes) in HA-R6 and RHA 397 were molecularly mapped to the lower end of linkage group 13, which encompasses a large R-gene cluster, and were designated as R _13a and R _13b, respectively. In the initial maps, SSR (simple sequence repeat) and InDel (insertion and deletion) markers revealed 2.8 and 8.2 cM flanking regions for R _13a and R _13b, respectively, linked with a common marker set of four co-segregating markers, ORS191, ORS316, ORS581, and ZVG61, in the distal side and one marker ORS464 in the proximal side. To identify new markers closer to the genes, sunflower RGC (resistance gene candidate) markers linked to the downy mildew R-gene Pl ₈ and located at the same region as R _13a and R _13b were selected to screen the two F₂ populations. The RGC markers RGC15/16 and a newly developed marker SUN14 designed from a BAC contig anchored by RGC251 further narrowed down the region flanking R _13a and R _13b to 1.1 and 0.1 cM, respectively. Both R _13a and R _13b are highly effective against all rust races tested so far. Our newly developed molecular markers will facilitate breeding efforts to pyramid the R ₁₃ genes with other rust R-genes and accelerate the development of rust-resistant sunflower hybrids in both confection and oilseed sunflowers.     

Identification of RAPD markers linked to a major blast resistance gene in rice

Rice blast, caused byPyricularia grisea, is a major production constraint in many parts of the world. The Korean rice variety Tongil showed high levels of resistance for about six years when widely planted under highly disease-conducive conditions, before becoming susceptible. Tongil was found to carry a single dominant gene, designatedPi-10t, conferring resistance to isolate 106 of the blast pathogen from the Philippines. We report here the use of bulked segregant RAPD analysis for rapid identification of DNA markers linked toPi-10t. Pooled DNA extracts from five homozygous blast-resistant (RR) and five susceptible (rr) BC₃F₂ plants, derived from a CO39 × Tongil cross, were analyzed by RFLP using 83 polymorphic probes and by RAPD using 468 random oligomers. We identified two RAPD markers linked to thePi-10t locus: RRF6 (3.8 ± 1.2 cM) and RRH18 (2.9 ± 0.9 cM). Linkage of these markers withPi-10t was verified using an F2 population segregating forPi-10t. The two linked RAPD markers mapped 7 cM apart on chromosome 5. Chromosomal regions surrounding thePi-10t gene were examined with additional RFLP markers to define the segment introgressed from the donor genome.Pi-10t is likely to be a new blast-resistance locus, because no other known resistance gene has been mapped on chromosome 5. These tightly linked RAPD markers could facilitate early selection of thePi-10t locus in rice breeding programmes.     

Development and validation of CAPS and AFLP markers for white rust resistance gene in<Emphasis Type="Italic"> Brassica juncea</Emphasis>

White rust, caused by Albugo candida, is a very serious disease in crucifers. In Indian mustard (Brassica juncea), it can cause a yield loss to the extent of 89.9%. The locus Ac2(t) controlling resistance to white rust in BEC-144, an exotic accession of mustard, was mapped using RAPD markers. In the present study, we developed: (1) a more tightly linked marker for the white rust resistance gene, using AFLP in conjunction with bulk segregant analysis, and (2) a PCR-based cleaved amplified polymorphic sequence (CAPS) marker for the closely linked RAPD marker, OPB06₁₀₀₀. The data obtained on 94 RILs revealed that the CAPS marker for OPB06₁₀₀₀ and the AFLP marker E-ACC/M-CAA₃₅₀ flank the Ac2(t) gene at 3.8 cM and 6.7 cM, respectively. Validation of the CAPS marker in two different F₂ populations of crosses Varuna × BEC-144 and Varuna × BEC-286 was also undertaken, which established its utility in marker-assisted selection (MAS) for white rust resistance. The use of both flanking markers in MAS would allow only 0.25% misclassification and thus provide greater efficiency to selection.Communicated by C. Möllers