Identification of molecular markers linked to the Agropyron elongatum-derived leaf rust resistance gene Lr24 in wheat

The objective of this study was to identify molecular markers linked to the wheat leaf rust resistance gene Lr24 derived from Agropyron elongatum (3DL/3Ag translocation). Two near isogenic lines (NILs), ‘Arina’ and Lr24/7 ^* “Arina”, were screened for polymorphism at the DNA level with 115 RFLP probes. Twenty-one of these probes map to the homoeologous group 3. In addition, 360 RAPD primers were tested on the NILs. Six RFLP probes showed polymorphism between the NILs, and 11 RAPD primers detected one additional band in the resistant NIL. The genetic linkage of the polymorphic markers with Lr24 was tested on a segregating F₂ population (150 plants) derived from a cross between the leaf rust resistant Lr24/7 ^* “Arina” and the susceptible spelt (Triticum spelta) variety ‘Oberkulmer’. All 6 RFLP markers were completely linked to Lr24: one was inherited as a codominant marker (PSR1205), one was in coupling phase (PSR1203) and 4 were in repulsion phase (PSR388, PSR904, PSR931, PSR1067) with Lr24. The localization of these probes on chromosome 3D was confirmed by nulli-tetrasomic analysis. Distorted genotypic segregation was found for the Codominant RFLP marker PSR1205. This distortion can be explained by the occurrence of hemizygous plants. One of the 11 RAPD markers (OPJ-09) also showed complete linkage to theLr24 resistance gene. The polymorphic RAPD fragment was cloned and sequenced. Specific primers were synthesized, and they produced an amplification product only in the resistant plants. This specific marker allows a reliable and rapid screening of a large number of genotypes in practical breeding. Analysis of 6 additional lines containing Lr24 revealed that 3 lines have a smaller chromosomal segment of A. elongatum than lines derived from ‘Agent’, a commonly used gene donor for the Lr24 resistance gene.     

Characterization of the Aspergillus niger pelB gene: structure and regulation of expression

Summary Nearly isogenic lines (NILs) of rice (Oryza sativa) differing at a locus conferring resistance to the pathogen Xanthomonas oryzae pv. oryzae were surveyed with 123 DNA markers and 985 random primers using restriction fragment length plymorphism (RFLP) and random amplified polymorphic DNA (RAPD) analysis. One chromosome 11 marker (RG103) detected polymorphism between the NILs that cosegregated with Xa21. All other chromosome 11 DNA markers tested were monomorphic between the NILs, localizing the Xa21 introgressed region to an 8.3 cM interval on chromosome 11. Furthermore, we identified two polymerase chain reaction (PCR) products (RAPD2148 and RAPD818) that detected polymorphisms between the NILs. Genomic sequences hybridizing with RAPD818, RAPD248 and RG103 were duplicated specifically in the Xa21 NIL. All three markers cosegregated with the resistance locus, Xa21, in a F₂ population of 386 progeny. Based on the frequency with which we recovered polymorphic Xa21-linked markers, we estimated the physical size of the introgressed region to be approximately 800 kb. This estimation was supported by physical mapping (using pulsed field gel electrophoresis) of the sequences hybridizing with the three Xa21-linked DNA markers. The results showed that the three Xa21-linked markers are physically close to each other, with one copy of the RAPD818 sequences located within 60 kb of RAPD248 and the other copy within 270 kb of RG103. None of the enzymes tested generated a DNA fragment that hybridized with all three of the markers indicating that the introgressed region containing the resistance locus Xa21 is probably larger than 270 kb.     

Molecular markers for wheat leaf rust resistance gene Lr41

Leaf rust, caused by Puccinia triticina Eriks., is an important foliar disease of common wheat (Triticum aestivum L.) worldwide. Pyramiding several major rust-resistance genes into one adapted cultivar is one strategy for obtaining more durable resistance. Molecular markers linked to these genes are essential tools for gene pyramiding. The rust-resistance gene Lr41 from T. tauschii has been introgressed into chromosome 2D of several wheat cultivars that are currently under commercial production. To discover molecular markers closely linked to Lr41, a set of near-isogenic lines (NILs) of the hard winter wheat cultivar Century were developed through backcrossing. A population of 95 BC₃F_2:6 NILs were evaluated for leaf rust resistance at both seedling and adult plant stages and analyzed with simple sequence repeat (SSR) markers using bulked segregant analysis. Four markers closely linked to Lr41 were identified on chromosome 2DS; the closest marker, Xbarc124, was about 1 cM from Lr41. Physical mapping using Chinese Spring nullitetrasomic and ditelosomic genetic stocks confirmed that markers linked to Lr41 were on chromosome arm 2DS. Marker analysis in a diverse set of wheat germplasm indicated that primers BARC124, GWM210, and GDM35 amplified polymorphic bands between most resistant and susceptible accessions and can be used for marker-assisted selection in breeding programs.     

Identification of a STS marker linked to the Aegilops speltoides-derived leaf rust resistance gene Lr28 in wheat

 A sequence-tagged-site (STS) marker is reported linked to Lr28, a leaf rust resistance gene in wheat. RAPD (random amplified polymorphic DNA) analysis of near-isogenic lines (NILs) of Lr28 in eight varietal backgrounds was carried out using random primers. Genomic DNA enriched for low-copy sequences was used for RAPD analysis to overcome the lack of reproducibility due to the highly repetitive DNA sequences present in wheat. Of 80 random primers tested on the enriched DNA, one RAPD marker distinguished the NILs and the donor parent from the susceptible recurrent parents. The additional band present in resistant lines was cloned, sequenced, and STS primers specific for Lr28 were designed. The STS marker (Indian patent pending: 380 Del98) was further confirmed by bulk segregation analysis of F₃ families. It was consistently present in the NILs, the resistant F₃ bulk and the resistant F₃ lines, but was absent in recurrent parents, the susceptible F₃ bulk and the susceptible F₃ lines. Received: 20 February 1998 / Accepted: 4 March 1998     

RFLP and RAPD mapping of the rice gm2 gene that confers resistance to biotype 1 of gall midge (Orseolia oryzae)

Gm2 is dominant gene conferring resistance to biotype 1 of gall midge (Orseolia oryzae Wood-Mason), the major dipteran pest of rice. The gene was mapped by restriction fragment length polymorphism (RFLP) analysis of a set of 40 recombinant inbred lines derived from a cross between the resistant variety Phalguna and the susceptible landrace ARC 6650. The gene is located on chromosome 4 at a position 1.3 cM from marker RG329 and 3.4 cM from RG476. Since the low (28%) polymorphism of this indica x indica cross hindered full coverage of the genome with RFLP markers, the mapping was checked by random amplified polymorphic DNA (RAPD)/bulked segregant analysis. Through the use of 160 RAPD primers, the number of polymorphic markers was increased from 43 to 231. Two RAPD primers amplified loci that co-segregated with resistance/susceptibility. RFLP mapping of these loci showed that they are located 0.7 cM and 2.0 cM from RG476, confirming the location of Gm2 in this region of chromosome 4. Use of these DNA markers will accelerate breeding for gall midge resistance by permitting selection of the Gm2 gene independently of the availability of the insect.     

SCAR,RAPD and RFLP markers linked to a dominant gene (Are) conferring resistance to anthracnose in common bean

Anthracnose, caused by the fungusColletotrichum lindemuthianum, is a severe disease of common bean (Phaseolus vulgaris L.) controlled, in Europe, by a single dominant gene,Are. Four pairs of near-isogenic lines (NILs) were constructed, in which theAre gene was introgressed into different genetic backgrounds. These pairs of NILs were used to search for DNA markers linked to the resistance gene. Nine molecular markers, five RAPDs and four RFLPs, were found to discriminate between the resistant and the susceptible members of these NILs. A backcross progeny of 120 individuals was analysed to map these markers in relation to theAre locus. Five out of the nine markers were shown to be linked to theAre gene within a distance of 12.0 cM. The most tightly linked, a RAPD marker, was used to generate a pair of primers that specifically amplify this RAPD (sequence characterized amplified region, SCAR).The research was supported by the CNRS and the Ministère Français de l'Education Nationale     

Molecular mapping of the py-1 gene for resistance to corky root rot (Pyrenochaeta lycopersici) in tomato

 We report the molecular mapping of the py-1 gene for resistance to corky root rot [Pyrenochaeta lycopersici (Schneider and Gerlach)] in tomato using RAPD and RFLP marker analysis. DNA from near-isogenic lines (NILs) of tomato differing in corky root rot resistance was screened with 575 random oligonucleotide primers to detect polymorphic DNAs linked to py-1. Three primers (OPW-04, OPC-02, OPG-19) revealed polymorphisms between the NILs. Twelve resistant and eight susceptible DNA pools derived from segregating F₃ families were used to confirm that the RAPD markers were linked to the py-1 gene. Two of the linked amplified fragments, corresponding to OPW-04 and OPC-02, were subsequently cloned and mapped on the tomato molecular linkage map as RFLPs. These clones were located between TG40 and CT31 on the short arm of chromosome 3. Further analysis with selected RFLP markers showed that 7% (8.8 cM) of chromosome 3 of the resistant line ‘Moboglan’ was introgressed from the L. peruvianum donor parent. Three RFLP markers (TG40, TG324, and TG479) from the introgressed part of chromosome 3 were converted to cleaved amplified polymorphism (CAP) markers for use in a polymerase chain reaction (PCR) assay. These PCR markers will allow rapid large-scale screening of tomato populations for corky root rot resistance. Received: 2 January 1998 / Accepted: 12 January 1998     

An RFLP marker in tomato linked to the Fusarium oxysporum resistance gene I2

Summary The locus, I2, which in tomato confers resistance against Fusarium oxysporum f. sp. lycopersici race 2, was introgressed into Lycopersicon esculentum from the wild species L. pimpinellifolium (P.I. 126915). We searched for restriction fragment length polymorphisms (RFLPs) between nearly isogenic lines (NILs) in clones that map to the region introgressed from the wild species. Since I2 maps to chromosome 11, we used DNA clones from this chromosome as hybridization probes to Southern blots containing bound DNA of the NILs digested with 23 restriction enzymes. Of the 14 chromosome 11 clones, 9 exhibited polymorphism. These clones were further hybridized to verification filters that contained DNA from resistant and susceptible L. esculentum varieties digested with the enzymes that gave the polymorphism. One clone, TG105, was found to be associated with I2; 19 susceptible lines showed a different RFLP with this probe than 16 resistant lines, including the original L. pimpinellifolium accession used as a source for the resistance gene. These results together with our mapping analysis indicate that TG105 is closely linked to the resistance gene.     

Chromosomal localization and molecular-marker tagging of the powdery mildew resistance gene (Lv) in tomato

We report the tagging of a powdery mildew [Leveillula taurica (Lév.) Arnaud.] resistance gene (Lv) in tomato using RAPD and RFLP markers. DNA from a resistant (cv Laurica) and a susceptible cultivar were screened with 300 random primers that were used to amplify DNA of resistant and susceptible plants. Four primers yielded fragments that were unique to the resistant line and linked to the resistance gene in an F₂ population. One of these amplified fragments, OP248, with a molecular weight of 0.7 kb, was subsequently mapped to chromosome 12, 1 cM away from CT134. Using RFLP markers located on chromosome 12, it was shown that approximately one half of chromosome 12 (about 42 cM), in the resistant variety is comprised of foreign DNA, presumably introgressed with the resistance gene from the wild species L. chilense. Further analysis of a backcross population revealed that the Lv gene lies in the 5.5-cM interval between RFLP markers, CT211 and CT219. As a prelude to map-based cloning of the Lv gene, we are currently enriching the density of markers in this region by a combination of RAPD primers and other techniques.     

Use of RFLP markers for the identification of alleles of the Pm3 locus conferring powdery mildew resistance in wheat (Triticum aestivum L.)

The objective of this study was to identify molecular markers linked to genes for resistance to powdery mildew (Pm) in wheat using a series of Chancellor near-isogenic-lines (NILs), each having one powdery mildew resistance gene. A total of 210 probes were screened for their ability to detect polymorphism between the NILs and the recurrent parent. One of these restriction fragment length polymorphism (RFLP) markers (Xwhs179) revealed polymorphism not only between the NILs for the Pm3 locus, but also among NILs possessing different alleles of the Pm3 locus. The location of the marker Xwhs179 was confirmed to be on homoeologous chromosome group 1 with the help of nullitetrasomic wheat lines. The linkage relationship between this probe and the Pm3 locus was estimated with double haploid lines derived from a cross between wheat cvs Club and Chul (Pm3b). The genetic distance was determined to be 3.3±1.9 cM.     

Tagging and combining bacterial blight resistance genes in rice using RAPD and RFLP markers

Four genes of rice,Oryza sativa L., conditioning resistance to the bacterial blight pathogenXanthomonas oryzae pv.oryzae (X. o. pv.oryzae), were tagged by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers. No recombinants were observed betweenxa-5 and RFLP marker lociRZ390, RG556 orRG207 on chromosome 5.Xa-3 andXa-4 were linked to RFLP locusXNpb181 at the top of chromosome 11, at distances of 2.3 cM and 1.7 cM, respectively. The nearest marker toXa-10, also located on chromosome 11, was the RAPD locusO07 ₂₀₀₀ at a distance of 5.3 cM. From this study, the conventional map [19, 28] and two RFLP linkage maps of chromosome 11 [14, 26] were partially integrated. Using the RFLP and RAPD markers linked to the resistance genes, we selected rice lines homozygous for pairs of resistance genes,Xa-4 +xa-5 andXa-4 +Xa-10. Lines carryingXa-4 +xa-5 andXa-4 +Xa-10 were evaluated for reaction to eight strains of the bacterial blight pathogen, representing eight pathotypes and three genetic lineages. As expected, the lines carrying pairs of genes were resistant to more of the isolates than their single-gene parental lines. Lines carryingXa-4 +xa-5 were more resistant to isolates of race 4 than were either of the parental lines (quantitative complementation). No such effects were seen forXa-4 +Xa-10. Thus, combinations of resistance genes provide broader spectra of resistance through both ordinary gene action expected and quantitative complementation.     

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.     

Use of RAPD markers to screen somatic hybrids between Solanum tuberosum and S. brevidens

Summary The identification of somatic hybrids between Solanum tuberosum and S. brevidens can be carried out using polymerase chain reaction (PCR) and arbitrary 10-mer primers to generate random amplified polymorphic DNA (RAPD) markers. Five commercial primers have been tested. Each primer directed the amplification of a genome-specific fingerprint for the fusion parents and S. brevidens. The size of the amplified DNA fragments ranged from 100 to 1800 base pairs. The somatic hybrids showed a combination of the parental banding profiles with four of the five primers surveyed, whereas regenerants from one of the parents had the same or a similar banding pattern to that of the parent. Thus RAPD markers provide a quick, simple and preliminary screening method for putative somatic hybrids.Abbreviations EDTA ethylenediaminetetraacetic acid, - PCR polymerase chain reaction - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphisms - TBE Tris-borate-EDTA buffer - Tris trizma base     

Mapping the Sw-5 locus for tomato spotted wilt virus resistance in tomatoes using RAPD and RFLP analyses

The Sw-5 locus confers dominant resistance to tomato spotted wilt virus (TSWV). To map the location and facilitate the identification of markers linked to Sw-5 we developed a pair of near-isogenic lines (NILs) and an F₂ Lycopersicon esculentum x L. pennellii population segregating for resistance to TSWV. DNA from the NILs was analyzed using 748 random 10-mer oligonucleotides to discern linked molecular markers using a random amplified polymorphic DNA (RAPD) approach. One random primer (GAGCACGGGA) was found to produce a RAPD band of about 2200 bp that demonstrates linkage to Sw-5. Data from co-segregation of resistance and restriction fragment length polymorphisms (RFLPs) in a F₂ interspecific population position Sw-5 between the markers CT71 and CT220 near the telomere of the long arm of chromosome 9.     

RAPD markers for constructing intraspecific tomato genetic maps

The existing molecular genetic maps of the tomato, Lycopersicon spp, are constructed based on isozyme and RFLP polymorphisms between tomato species. These maps are useful for certain applications but have few markers that exhibit sufficient polymorphisms for intraspecific analysis and manipulations within the cultivated tomato. The purpose of this study was to investigate the relative potential of RAPD technology, as compared to isozymes and RFLPs, to generate polymorphic DNA markers within cultivated tomatoes. Sixteen isozymes and 25 RFLP clones that were known to detect polymorphism between L. esculentum and L. pennellii, and 313 random oligonucleotide primers were examined. None of the isozymes and only four of the RFLP clones (i.e., 16%) revealed polymorphism between the cultivated varieties whereas up to 63% of the RAPD primers detected one or more polymorphic DNA fragments between these varieties. All RAPD primers detected polymorphism between L. esculentum and L. pennellii genotypes. These results clearly indicate that RAPD technology can generate sufficient genetic markers exploiting sequence differences within cultivated tomatoes to facilitate construction of intraspecific genetic maps.Abbreviations RFLP restriction fragments length polymorphism - RAPD random amplified polymorphic DNA - PCR polymerase chain reaction - QTLs quantitative trait loci     

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.     

Molecular characterization of RAPD and SCAR markers linked to the Tm-1 locus in tomato

We have cloned and sequenced six RAPD fragments tightly linked to the Tm-1 gene which confers tomato mosaic virus (ToMV) resistance in tomato. The terminal ten bases in each of these clones exactly matched the sequence of the primer for amplifying the corresponding RAPD marker, except for one in which the 5-endmost two nucleotides were different from those of the primer. These RAPD clones did not cross-hybridize with each other, suggesting that they were derived from different loci. From Southern-hybridization experiments, five out of the six RAPD clones were estimated to be derived from middle- or high-repetitive sequences, but not from any parts of the ribosomal RNA genes (rDNA), which are known to be tightly linked with the Tm-1 locus. The remaining clone appeared to be derived from a DNA family consisting of a few copies. These six RAPD fragments were converted to sequence characterized amplified region (SCAR) markers, each of which was detectable using a pair of primers having the same sequence as that at either end of the corresponding RAPD clone. All pairs of SCAR primers amplified distinct single bands whose sizes were the same as those of the RAPD clones. In four cases, the SCAR markers were present in the line with Tm-1 but absent in the line without it, as were the corresponding RAPD markers. In the two other cases, the products of the same size were amplified in both lines. When these SCAR products were digested with different restriction endonucleases which recognize 4-bp sequences, however, polymorphisms in fragment length were found between the two lines. These co-dominant markers are useful for differentiating heterozygotes from both types of homozygote.     

Tagging genes for blast resistance in rice via linkage to RFLP markers

Summary Both Pi-2(t) and Pi-4(t) genes of rice confer complete resistance to the blast fungal pathogen Pyricularia oryzae Cav. As economically important plant genes, they have been recently characterized phenotypically, yet nothing is known about their classical linkage associations and gene products. We report here the isolation of DNA markers closely linked to these blast resistance genes in rice. The DNA markers were identified by testing 142 mapped rice genomic clones as hybridization probes against Southern blots, consisting of DNA from pairs of nearly isogenic lines (NILs) with or without the target genes. Chromosomal segments introgressed from donor genomes were distinguished by restriction fragment length polymorphisms (RFLPs) between the NILs. Linkage associations of the clones with Pi-2(t) and Pi4(t) were verified using F₃ segregating populations of known blast reaction. Cosegregation of the resistant genotype and donor-derived allele indicated the presence of linkage between the DNA marker and a blast resistance gene. RFLP analysis showed that Pi-2(t) is closely linked to a single-copy DNA clone RG64 on chromosome 6, with a distance of 2.8+1.4(SE) cMorgans. Another blast resistance gene, Pi-4(t), is 15.3+4.2(SE) cMorgans away from a DNA clone RG869 on chromosome 12. These chromosomal regions can now be examined with additional markers to define the precise locations of Pi-2(t) and Pi-4(t). Tightly linked DNA markers may facilitate early selection for blast resistance genes in breeding programs. These markers may also be useful to map new genes for resistance to blast isolates. They may ultimately lead to the cloning of those genes via chromosome walking. The gene tagging approach demonstrated in this paper may apply to other genes of interest for both monogenic and polygenic traits.     

Molecular identification of powdery mildew resistance genes in common wheat (Triticum aestivum L.)

RFLP markers for the wheat powdery mildew resistance genes Pm1 and Pm2 were tagged by means of near-isogenic lines. The probe Whs178 is located 3 cM from the Pm1 gene. For the powdery mildew resistance gene Pm2, two markers were identified. The linkage between the Pm2 resistance locus and one of these two probes was estimated to be 3 cM with a F₂ population. Both markers can be used to detect the presence of the corresponding resistance gene in commercial cultivars. Bulked segregant analysis was applied to identify linkage disequillibrium between the resistance gene Pm18 and the abovementioned marker, which was linked to this locus at a distance of 4 cM. Furthermore, the RAPD marker OPH-11₁₉₀₀ (5-CTTCCGCAGT-3) was selected with pools created from a population segregating for the resistance of Trigo BR 34. The RAPD marker was mapped about 13 cM from this resistance locus.     

Evaluation of RFLP and RAPD markers in a comparison of Brassica napus breeding lines

RFLP and RAPD markers were evaluated and compared for their ability to determine genetic relationships in a set of three B. napus breeding lines. Using a total of 50 RFLP and 92 RAPD markers, the relatedness between the lines was determined. In total, the RFLP and the RAPD analysis revealed more than 500 and 400 bands, respectively. The relative frequencies of loci with allele differences were estimated from the band data. The RFLP and RAPD marker sets detected very similar relationships among the three lines, consistent with known pedigree data. Bootstrap analyses showed that the use of approximately 30 probes or primers would have been sufficient to achieve these relationships. This indicates that RAPD markers have the same resolving power as RFLP markers when used on exactly the same set of B. napus genotypes. Since RAPD markers are easier and quicker to use, these markers may be preferred in applications where the relationships between closely-related breeding lines are of interest. The use of RAPD markers in fingerprinting applications may, however, not be warranted, and this is discussed in relation to the reliability of RAPD markers.