Construction of a BAC contig containing the xa5 locus in rice

 The recessive gene xa5 confers resistance to bacterial blight in rice. To generate a physical map of the xa5 locus, three RFLP markers RG556, RG207 and RZ390, closely linked to xa5, were used to screen a rice bacterial artificial chromosome (BAC) library. The identified overlapping BAC clones formed two small contigs which were extended to both sides by chromosome walking. The final physical map consisted of 14 BAC clones and covered 550 kb. Genetic analysis with an F₂ population showed that two RFLP markers 28N22R and 40F20R, derived from the BAC clones in the contig, flanked the xa5 locus. To further delimit the location of the xa5 locus, RFLP markers RG556 and RG207 were converted to sequence tagged sites and used to perform genetic analysis. The results indicated that the xa5 locus was most likely located between RG207 and RG556. Among the BAC clones in the contig, one clone, 44B4, hybridized to both RG207 and RG556. This suggests that BAC clone 44B4 carried the xa5 locus. Received: 12 January 1998 / Accepted: 27 May 1998     

AFLP markers tightly linked to the aluminum-tolerance gene Alt3 in rye (Secale cereale L.)

Rye (Secale cereale L.) is considered to be the most aluminum (Al)-tolerant species among the Triticeae. It has been suggested that aluminum tolerance in rye is controlled by three major genes (Alt genes) located on rye chromosome arms 3RL, 4RL, and 6RS, respectively. Screening of an F₆ rye recombinant inbred line (RIL) population derived from the cross between an Al-tolerant rye (M39A-1–6) and an Al-sensitive rye (M77A-1) showed that a single gene controls aluminum tolerance in the population analyzed. In order to identify molecular markers tightly linked to the gene, we used a combination of amplified fragment length polymorphism (AFLP) and bulked segregant analysis techniques to evaluate the F₆ rye RIL population. We analyzed approximately 22,500 selectively amplified DNA fragments using 204 primer combinations and identified three AFLP markers tightly linked to the Alt gene. Two of these markers flanked the Alt locus at distance of 0.4 and 0.7 cM. Chromosomal localization using cloned AFLP and a restriction fragment length polymorphism (RFLP) marker indicated that the gene was on the long arm of rye chromosome 4R. The RFLP marker (BCD1230) co-segregated with the Alt gene. Since the gene is on chromosome 4R, the gene was designated as Alt3. These markers are being used as a starting point in the construction of a high resolution map of the Alt3 region in rye. Received: 29 March 2000 / Accepted: 9 July 2001     

RFLP mapping of the genes controlling hybrid breakdown in rice (Oryza sativa L.)

 Complementary recessive genes hwd1 and hwd2 controlling hybrid breakdown (weakness of F₂ and later generations) were mapped in rice using RFLP markers. These genes produce a plant that is shorter and has fewer tillers than normal plants when the two loci have only one or no dominant allele at both loci. A cultivar with two dominant alleles at the hwd1 locus and a cultivar with two dominant alleles at the hwd2 locus were crossed with a double recessive tester line. Linkage analysis was carried out for each gene independently in two F₂ populations derived from these crosses. hwd1 was mapped on the distal region of rice genetic linkage map for chromosome 10, flanked by RFLP markers C701 and R2309 at a distance of 0.9 centiMorgans (cM) and 0.6 cM, respectively. hwd2 was mapped in the central region of rice genetic linkage map for chromosome 7, tightly linked with 4 RFLP markers without detectable recombination. The usefulness of RFLP mapping and map information for the genes controlling reproductive barriers are discussed in the context of breeding using diverse rice germplasm, especially gene introduction by marker-aided selection.     

Extended physical maps and a consensus physical map of the homoeologous group-6 chromosomes of wheat (Triticum aestivum L. em Thell.)

Extended physical maps of chromosomes 6A, 6B and 6D of common wheat (Triticum aestivum L. em Thell., 2n=6x=42, AABBDD) were constructed with 107 DNA clones and 45 homoeologous group-6 deletion lines. Two-hundred and ten RFLP loci were mapped, including three orthologous loci with each of 34 clones, two orthologous loci with each of 31 clones, one locus with 40 clones, two paralogous loci with one clone, and four loci, including three orthologs and one paralog, with one clone. Fifty five, 74 and 81 loci were mapped in 6A, 6B and 6D, respectively. The linear orders of the mapped orthologous loci in 6A, 6B and 6D appear to be identical and 65 loci were placed on a group-6 consensus physical map. Comparison of the consensus physical map with eight linkage maps of homoeologous group-6 chromosomes from six Triticeaespecies disclosed that the linear orders of the loci on the maps are largely, if not entirely, conserved. The relative distributions of loci on the physical and linkage maps differ markedly, however. On most of the linkage maps, the loci are either distributed relatively evenly or clustered around the centromere. In contrast, approximately 90% of the loci on the three physical maps are located either in the distal one-half or the distal two-thirds of the six chromosome arms and most of the loci are clustered in two or three segments in each chromosome. Received: 19 April 1999 / Accepted: 28 July 1999     

Chromosome landing at the Mla locus in barley (Hordeum vulgare L.) by means of high-resolution mapping with AFLP markers

 The complex Mla locus of barley determines resistance to the powdery mildew pathogen Erysiphe graminis f. sp. hordei. With a view towards gene isolation, a population consisting of 950 F₂ individuals derived from a cross between the near-isogenic lines ‘P01’ (Mla1) and ‘P10’ (Mla12) was used to construct a high-resolution map of the Mla region. A fluorescence-based AFLP technique and bulked segregant analysis were applied to screen for polymorphic, tightly linked AFLP markers. Three AFLP markers were selected as suitable for a chromosome-landing strategy. One of these AFLP markers and a closely linked RFLP marker were converted into sequence-specific PCR markers. PCR-based screening of approximately 70 000 yeast artificial chromosome (YAC) clones revealed three identical YACs harbouring the Mla locus. Terminal insert sequences were obtained using inverse PCR. The derived STS marker from the right YAC end-clone was mapped distal to the Mla locus. Received: 17 July 1998 / Accepted: 9 August 1998     

RFLP-based mapping of three mutant loci in rye (Secale cereale L.) and their relation to homoeologous loci within the Gramineae

 Three mutant loci of rye determining absence of ligules (al), waxless plant (wa1) and waxy endosperm (Wx) characters were mapped in a single F₂ population, comprising 84 individual plants. The three loci could be clearly tagged in relation to 7 (al on chromosome 2R), 4 (wa1 on chromosome 7R) or 6 (Wx on chromosome 4R) RFLP markers. The mapping data are compared with existing data for homoeologous regions containing equivalent mutants of wheat, barley, rice and maize. It is shown that the loci analysed are highly conserved across the cereal species, including rye. Received: 14 March 1997 / Accepted: 21 March 1997     

A molecular linkage map of rye

A genetic map of six chromosomes of rye, (all of the rye chromosomes except for 2R), was constructed using 77 RFLP and 12 RAPD markers. The map was developed using an F₂ population of 54 plants from a cross between two inbred lines. A rye genomic library was constructed as a source of clones for RFLP mapping. Comparisons were made between the rye map and other rye and wheat maps by including additional probes previously mapped in those species. These comparisons allowed (1) chromosome arm orientation to the linkage groups to be given, (2) the corroboration of several evolutionary translocations between rye chromosomes and homoeologous chromosomes of wheat; (3) an increase in the number of available markers for target regions of rye that show colinearity with wheat. Inconsistencies in the location of markers between the wheat and rye maps were mostly detected by multi-copy probes.     

Molecular linkage mapping in rye (Secale cereale L.)

A rye linkage map containing clones from rye, wheat, barley, oat and rice genomic and cDNA libraries, known-function genes and microsatellite markers, was created using an F₂ population consisting of 110 F₂-derived F₃ families. Both co-dominant and dominant markers were added to the map. Of all probes screened, 30.8% were polymorphic, and of those polymorphic 79.3% were mapped. The current map contains 184 markers present in all seven linkage groups covering only 727.3 cM. This places a marker about every 3.96 cM on average throughout the map; however, large gaps are still present. The map contains 60 markers that have been integrated from previous rye maps. Surprisingly, no markers were placed between the centromere and C1–1RS in the short arm of 1R. The short arm of chromosome 4 also lacked an adequate number of polymorphic markers. The population showed a remarkable degree of segregation distortion (72.8%). In addition, the genetic distance observed in rye was found to be very different among the maps created by different mapping populations. Received: 10 January 2000 / Accepted: 26 May 2000     

Mapping of meiotic genes in rye (Secale cereale L.): Localization of sy19 mutation, impairing homologous synapsis, by means of isozyme and microsatellite markers

The sy19 mutation, which impairs the homology of meiotic chromosome synapsis in rye, were mapped using a specially created F₂ population by means of isozyme Acph1 locus and microsatellite (SSR) markers. The sy19 gene was localized in the chromosome 7R in the pericentromeric region of long arm based on the linked inheritance with the Acph1 locus. The locus was linked with five rye SSR markers, with the Xrems1234 locus being located closest to the sy19 gene (6.4 cM). The genetic map of the analyzed chromosome 7R region includes ten markers and the sy19 locus. A possible function of the Sy1 and Sy19 genes based on the data on comparative genomics is discussed.     

High-density physical maps reveal that the dominant male-sterile gene Ms3 is located in a genomic region of low recombination in wheat and is not amenable to map-based cloning

In wheat it is essential to know whether a gene is located in a high or low recombination region of the genome before initiating a map-based cloning approach. The objective of this study was to explore the potential feasibility of map-based cloning of the dominant male-sterile gene Ms3 of wheat. High-density physical maps of the short arms of the group-5 chromosomes (5AS, 5BS, and 5DS) of Triticum aestivum L. were constructed by mapping 40 DNA markers on a set of 17 homozygous deletion lines. One hundred RFLP loci were mapped: 35 on 5AS, 37 on 5BS, and 28 on 5DS. A consensus physical map was colinearly aligned with a consensus genetic map of the group-5 short arms. Sixteen of the 17 markers in the consensus genetic map encompass a genetic distance of 25 cM and correspond to the distal region (FL 0.56–0.97) of the consensus physical map. Two rice probes, RG463 and RG901, previously identified to be linked to markers CDO344 and CDO749 (group-5 short arm of wheat), respectively, in the genetic map of rice chromosome 12, map between FL 0.56 and 0.63 in the consensus map. Thus at least a part of the group-5 short arm is homoeologous to a region of chromosome 12 of rice. The genetic map of chromosome arm 5AS was constructed using a population of 139 BC₁ plants derived from a cross between the euploid wheat ”Chris” carrying a dominant male-sterile gene Ms3 and a disomic substitution line in which chromosome 5A of T. aestivum cv Chinese Spring was substituted by chromosome 5A from Triticum turgidum ssp. dicoccoides. The map has a genetic length of 53.4 cM with 11 DNA markers. The initial map showed that the gene Ms3 cosegregated with three markers, WG341, BCD1130 and CDO677. High-resolution mapping using an additional 509 BC₁ plants indicated that the marker WG341 was closely linked to Ms3 at a genetic distance of 0.8 cM. The Ms3 was mapped physically in the region spanning 40% of the arm length from the centromere of 5AS. Therefore, map-based cloning of the Ms3 is not feasible, although WG341 can be used as a useful tag for the Ms3 gene for breeding purposes. Received: 12 December 2000 / Accepted: 26 January 2001     

Isolation and FISH mapping of Yeast Artificial Chromosomes (YACs) encompassing an allele of the Gm2 gene for gall midge resistance in rice

 Ten yeast artificial chromosomes (YACs) spanning the Gm2 locus have been isolated by screening high-density filters containing a total of approximately 7000 YAC (representing six genome equivalents) clones derived from a japonica rice, Nipponbare. The screening was done with five RFLP markers flanking a gall midge resistance gene, Gm2, which was previously mapped onto chromosome 4 of rice. This gene confers resistance to biotype 1 and 2 of gall midge (Orseolia oryzae), a major insect pest of rice in South and Southeast Asia. The RFLP markers RG214, RG329 and F8 hybridized with YAC Y2165. Two overlapping YAC clones (Y5212 and Y2165) were identified by Southern hybridization, with Gm2-flanking RFLP markers, and their inserts isolated. The purified YACs and RFLP markers flanking Gm2 were labeled and physically mapped by the fluorescence in situ hybridization (FISH) technique. All of them mapped to the long arm of chromosome 4 of the resistant variety of rice, ‘Phalguna’, confirming the previous RFLP mapping data. Received: 15 December 1997 / Accepted: 5 March 1998     

The Pik m gene, conferring stable resistance to isolates of Magnaporthe oryzae , was finely mapped in a crossover-cold region on rice chromosome 11

The Pik ^m gene in rice confers a high and stable resistance to many isolates of Magnaporthe oryzae collected from southern China. This gene locus was roughly mapped to the long arm of rice chromosome 11 with restriction fragment length polymorphic (RFLP) markers in the previous study. To effectively utilize the resistance, a linkage analysis was performed in a mapping population consisting of 659 highly susceptible plants collected from four F₂ populations using the publicly available simple sequence repeat (SSR) markers. The result showed that the locus was linked to the six SSR markers and defined by RM254 and RM144 with ≈13.4 and ≈1.2 cM, respectively. To fine map this locus, additional 10 PCR-based markers were developed in a region flanked by RM254 and RM144 through bioinformatics analysis (BIA) using the reference sequence of cv. Nipponbare. The linkage analysis with these 10 markers showed that the locus was further delimited to a 0.3-cM region flanked by K34 and K10, in which three markers, K27, K28, and K33, completely co-segregated with the locus. To physically map the locus, the Pik ^m -linked markers were anchored to bacterial artificial chromosome clones of the reference cv. Nipponbare by BIA. A physical map spanning ≈278 kb in length was constructed by alignment of sequences of the clones anchored by BIA, in which only six candidate genes having the R gene conserved structure, protein kinase, were further identified in an 84-kb segment.     

Chromosome landing at the Arabidopsis TORNADO1 locus using an AFLP-based strategy

 The Arabidopsis tornado1 (trn1) mutation causes severe dwarfism combined with twisted growth of all organs. We present a chromosome landing strategy, using amplified restriction fragment length polymorphism (AFLP) marker technology, for the isolation of the TRN1 gene. The recessive trn1 mutation was identified in a C24 transgenic line and is located 5 cM from a T-DNA insertion. We mapped the TRN1 locus to the bottom half of chromosome 5 relative to visible and restriction fragment length polymorphism (RFLP) markers. Recombinant classes within a 3-cM region around TRN1 were used to build a high-resolution map in this region, using the AFLP technique. Approximately 300 primer combinations have been used to test about 26 000 fragments for polymorphisms. Seventeen of these AFLP markers were identified in the 3-cM region around TRN1. These markers were mapped within this region using individual recombinants. Four of these AFLP markers co-segregate with TRN1 whereas one maps at one recombinant below TRN1. We isolated and cloned three of these AFLP markers. These markers identified two yeast artificial chromosome (YAC) clones, containing the RFLP marker above and the AFLP marker below TRN1, demonstrating that these YACs span the TRN1 locus and that chromosome landing has been achieved, using an AFLP-based strategy. Received: 25 April 1996 / Accepted: 26 June 1996     

A high-resolution linkage map of the vicinity of the rice submergence tolerance locus Sub1

Resistance to submergence stress is an important breeding objective in areas where rice cultivars are subjected to complete inundation for a week or more. The present study was conducted to develop a high-resolution map of the region surrounding the submergence tolerance gene Sub1 in rice, which derives from the Indian cultivar FR13A. Submergence screening of 8-day-old plants of F₃ families kept for 14 days submerged in 60 cm of water allowed an accurate classification of Sub1 phenotypes. Bulked segregant analysis was used to identify AFLP markers linked to Sub1. A population of 2950 F₂ plants segregating for Sub1 was screened with two RFLP markers flanking the Sub1 locus, 2.4 and 4.9 cM away. Submergence tolerance was measured in the recombinant plants, and AFLP markers closely linked to Sub1 were mapped. Two AFLP markers cosegregated with Sub1 in this large population, and other markers were localized within 0.2 cM of Sub1. The high-resolution map should serve as the basis for map-based cloning of this important locus, as it will permit the identification of BAC clones spanning the region. Received: 15 December 1999 / Accepted: 18 February 2000     

Marker enrichment and high-resolution map of the segment of potato chromosome VII harbouring the nematode resistance gene Gro1

The dominant allele Gro1 confers on potato resistance to the root cyst nematode Globodera rostochiensis. The Gro1 locus has been mapped to chromosome VII on the genetic map of potato, using RFLP markers. This makes possible the cloning of Gro1 based on its map position. As part of this strategy we have constructed a high-resolution genetic map of the chromosome segment surrounding Gro1, based on RFLP, RAPD and AFLP markers. RAPD and RFLP markers closely linked to Gro1 were selected by bulked segregant analysis and mapped relative to the Gro1 locus in a segregating population of 1105 plants. Three RFLP and one RAPD marker were found to be inseparable from the Gro1 locus. Two AFLP markers were identified that flanked Gro1 at genetic distances of 0.6 cM and 0.8 cM, respectively. A genetic distance of 1 cM in the Gro1 region corresponds to a physical distance of ca. 100 kb as estimated by long-range restriction analysis. Marker-assisted selection for nematode resistance was accomplished in the course of constructing the high-resolution map. Plants carrying the resistance allele Gro1 could be distinguished from susceptible plants by marker assays based on the polymerase chain reaction (PCR).     

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     

A genetic and physical map of the region containing PLASTOCHRON1, a heterochronic gene,in rice ( Oryza sativa L.)

The rice heterochronic gene plastochron1, pla1, shows shorter plastochron and ectopic expression of the vegetative program during the rice reproductive phase resulting in aberrant panicle formation. A genetic and physical map was constructed to isolate the causal gene for the pla1 syndrome. Small-scale mapping was carried out to determine the approximate map position of the pla1 locus, and then a high-resolution genetic map was made for pla1-1, one of the pla1 alleles, using an F₂ population comprising 578 pla1-1 homozygous plants. In a high-resolution genetic map, the pla1-1 locus was found to map between RFLP markers C961 and R1738A on chromosome 10, within a 3.6-cM genetic distance. A physical map encompassing the pla1-1 locus was constructed by overlapping Bacterial Artificial Chromosome (BAC) clones through chromosome walking. PCR-based RFLP markers from BAC-end clones were developed and mapped relative to the pla1 locus. Physical map construction using BAC clones indicated that a BAC clone, B44A10 (167-kb), contained the pla1 locus within 74-kb corresponding to a 0.52-cM genetic distance. Gene prediction of 74-kb region carrying the pla1 locus suggested several candidate genes for the pla1 gene. Identification of a candidate gene for pla1 will be made by sequence analysis of allele variation and cDNA screening.     

RFLP mapping of genes affecting plant height and growth habit in rye

Summary RFLP mapping of chromosome 5R in the F₃ generation of a rye (Secale cereale L.) cross segregating for gibberellic acid (GA₃)-insensitive dwarfness (Ct2/ct2) and spring growth habit (Sp1/sp1) identified RFLP loci close to each of these agronomically important genes. The level of RFLP in the segregating population was high, and thus allowed more than half of the RFLP loci to be mapped, despite partial homozygosity in the parental F₂ plant. Eight further loci were mapped in an unrelated F₂ rye population, and a further two were placed by inference from equivalent genetic maps of related wheat chromosomes, allowing a consensus map of rye chromosome 5R, consisting of 29 points and spanning 129 cM, to be constructed. The location of the ct2 dwarfing gene was shown to be separated from the segment of the primitive 4RL translocated to 5RL, and thus the gene is probably genetically unrelated to the major GA-insensitive Rht genes of wheat located on chromosome arms 4BS and 4DS. The map position of Sp1 is consistent both with those of wheat Vrn1 and Vrn3, present on chromosome arms 5AL and 5DL, respectively, and with barley Sh2 which is distally located on chromosome arm 7L (= 5HL).     

Genetic mapping of Dn7, a rye gene conferring resistance to the Russian wheat aphid in wheat

The Russian wheat aphid is a significant pest problem in wheat and barley in North America. Genetic resistance in wheat is the most effective and economical means to control the damage caused by the aphid. Dn7 is a rye gene located on chromosome 1RS that confers resistance to the Russian wheat aphid. The gene was previously transferred from rye into a wheat background via a 1RS/1BL translocation. This study was conducted to genetically map Dn7 and to characterize the type of resistance the gene confers. The resistant line '94M370' was crossed with a susceptible wheat cultivar that also contains a pair of 1RS/1BL translocation chromosomes. The F₂ progeny from this cross segregated for resistance in a ratio of 3 resistant: 1 susceptible, indicating a single dominant gene. One-hundred and eleven RFLP markers previously mapped on wheat chromosomes 1A, 1B and 1D, barley chromosome 1H and rye chromosome 1R, were used to screen the parents for polymorphism. A genetic map containing six markers linked to Dn7, encompassing 28.2 cM, was constructed. The markers flanking Dn7 were Xbcd1434 and XksuD14, which mapped 1.4 cM and 7.4 cM from Dn7, respectively. Dn7 confers antixenosis, and provides a higher level of resistance than that provided by Dn4. The applications of Dn7 and the linked markers in wheat breeding are discussed.Communicated by J. Dvorak     

Towards an integrated linkage map of common bean. 4. Development of a core linkage map and alignment of RFLP maps

 Three RFLP maps, as well as several RAPD maps have been developed in common bean (Phaseolus vulgaris L.). In order to align these maps, a core linkage map was established in the recombinant inbred population BAT93×Jalo EEP558 (BJ). This map has a total length of 1226 cM and comprises 563 markers, including some 120 RFLP and 430 RAPD markers, in addition to a few isozyme and phenotypic marker loci. Among the RFLPs mapped were markers from the University of California, Davis (established in the F₂ of the BJ cross), University of Paris-Orsay, and University of Florida maps. These shared markers allowed us to establish a correspondence between the linkage groups of these three RFLP linkage maps. In total, the general map location (i.e., the linkage group membership and approximate location within linkage groups) has been determined for some 1070 markers. Approaches to align this core map with other current or future maps are discussed. Received: 10 March 1998 / Accepted: 22 April 1998