PCR–RFLP assays for discerning three weevil stem feeders (Ceutorhynchus spp.) (Col.: Curculionidae) on garlic mustard (Alliaria petiolata)

Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) assays were developed to rapidly and cost-effectively differentiate the morphologically indistinguishable larval stages of three species, Ceutorhynchus alliariae, C. roberti and C. scrobicollis, proposed for the biological control of Alliaria petiolata. A PCR–RFLP using TaqαI restriction enzyme can differentiate the three target species only, while a PCR–RFLP double digestion using the restriction enzymes AluI and TaqαI should enable, based on virtual digestion, distinguishing them from 12 other Ceutorhynchus species associated with A. petiolata.     

Restriction fragment length polymorphism at the HLA-C locus

We have used the HLA-C-specific DNA probe pC250 to investigate restriction fragment length polymorphism (RFLP) at the HLA-C locus. Genomic Southern blot hybridization included DNA prepared from a panel of homozygous typing cells representing serological specificities Cw1 to Cw8 and also from samples representing Cw blanks. Although many restriction nucleases failed to reveal any polymorphism, RFLPs were evident with Taq I, Pvu II, Bst XI, Nde 1, and Nci I in addition to the previously reported Eco RI. In the case of Bst XI, a unique RFLP defined a subset of serologically defined Cw blanks. Comparison of RFLP sizes with restriction fragment lengths obtained from the known HLA-Cw3 gene sequence permitted the localization of intragenic C locus RFLLs and the identification of a variable Taq I site in the second intron, a variable Nci I site near the end of the fourth exon, and a variable Pvu lI site in the fifth intron.     

Identification of an RFLP marker tightly linked to theHt1 gene in maize

Summary We have identified tight linkage of an RFLP marker to theHt1 gene of maize that confers resistance to the fungal pathogenHelminthosporium turcicum race 1. This was accomplished by the use of four pairs of near isogenic lines (NILs; B73, A619, W153R, and CM105), each differing by the presence or the absence of the geneHt1. SinceHt1 maps to chromosome 2, 26 clones already mapped to this chromosome were labeled and probed against Southern blots of these NILs DNA digested with three restriction enzymes:EcoRI,BamHI, andHindIII. Six markers exhibited an RFLP for at least one pair of NILs. Presumptive linkage was further tested by analyzing the segregation of five of the six markers (one was monomorphic in the cross studied) and resistance toH. turcicum race 1 on 95 F₂ individuals from the cross DF20 × LH146Ht. The results indicate a tight linkage between one of the DNA markers,UMC150B, and theHt1 gene.     

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.     

High-resolution mapping of loci conferring resistance to sugarcane mosaic virus in maize using RFLP, SSR, and AFLP markers

Sugarcane mosaic virus (SCMV) is one of the most important virus diseases of maize in Europe. Genetic analysis on backcross five (BC5) progeny derived from the cross FAP1360A (resistant) × F7 (susceptible) confirmed that at least two dominant genes, Scm1 and Scm2, are required for resistance to SCMV in the progeny of this cross. With the aid of RFLP and SSR marker analyses, Scm1 was mapped in the region of 8.7 cM – between the nucleolus organizer region (nor) and RFLP marker bnl6.29 on the short arm of chromosome 6, while Scm2 was mapped to an interval of 26.8 cM flanked by the RFLP markers umc92 and umc102 near the centromere region of chromosome 3. Both chromosome regions were further enriched for AFLP markers by successful application of a bulked segregant analysis to this oligogenic trait. A total of 23 linked AFLP markers were identified, clustered in chromosome regions adjacent to either Scm1 or Scm2. Seven AFLP markers linked to Scm1 resided within the nor-bnl6.29 interval, and one of them, E3M8-1, showed no recombination with Scm1. Three AFLP markers linked to Scm2 are located between umc92 and umc102. Received: 13 October 1998 / Accepted: 18 January 1999     

DQ α and β RFLP reveals the composition of the DQ molecule recognized by T-cell clones

Pst I RFLP, revealed with DQ and DQ probes, was compared with Taq I RFLP using a panel of DR-homozygous cell lines and HLA-typed family members. Taq I patterns, characteristic for each DR-associated DQ and allelic forms, were recognized in the homozygous state and then proven to segregate in the heterozygous members of informative families. The presence of both specific and chains was found to be necessary to form the type of DQ molecule specifically recognized by two alloreactive T-cell clones. Particular and associations also seem to be responsible for some Dw splits of the DRw6-positive cells. Taq I RFLP analysis may be more complex than the Pst I analysis, but is certainly more informative and complete, considering the type of information we were seeking by performing these types of experiments.Abbreviations used in this paper BSA bovine serum albumin - GLO glyoxalase - kb kilobase(s) - LCL lymphoblastoid cell line - MHC major histocompatibility complex - PBL peripheral blood lymphocyte - PLT primed lymphocyte test - RFLP restriction fragment length polymorphism - SDS sodium dodecyl sulfate - SSC standard sodium citrate - SSCP sodium, sodium citrate, sodium phosphate - TBE Tris-borate, boric acid, ethylenediaminetetraacetate (EDTA) - TCGF T-cell growth factor     

Novel detection of restriction fragment length polymorphisms in the human major histocompatibility complex

Cosmid genomic DNA clones have been used as hybridization probes in genomic Southern blot analysis to define restriction fragment length polymorphisms (RFLPs) in the major histocompatibility complex (MHC). Using 14 different enzymes and three overlapping cosmid clones we have detected six RFLPs in a 100 kilobase (kb) segment of DNA in the class III region extending centromeric of theTNFA gene towardHLA-DR. Four of the five RFLPs, defined using the enzymesTaqI,Rsa I,Hinc II, andHind III, and detected by the cosmid clone cosM7B, map to a 29 kb segment of DNA that includes all of the recently described G2 (BAT2) gene and a large portion of the 3 end of the G3 (BAT3) gene. The different RFLP variants were established by analyzing the DNA from three informative families and a panel of 51HLA-homozygous typing cell lines. CosM7B detectsTaq I variants of 4.3 kb, and 2.9 kb or 2.8 kb, Rsa I variants of 2.9 kb or 2.4 kb,Hinc II variants of 5.8 kb or 3.8 kb and 1.4 kb, and aHind III variant of 4.8 kb, while cosOT2 detects Taq I variants of 4.5 kb or 4 kb. The distribution of theRsa 1, Hinc II and Taq I RFLPs detected by cosM7B, and theTaq I RFLP detected with cosOT2, within the panel of cell line DNAs was assessed by Southern blotting. The 4.3 kbTaq I variant was observed in only one cell line with the extended haplotypeHLA-A29, C-, B44, SC30, DR4. The other RFLPs, however, occurred much more frequently. The 2.8 kb Taq I variant was observed in 20 % of haplotypes, the 2.9 kbRsa I variant was observed in 42% of haplotypes, and the 5.8 kbHinc I variant was observed in 12 % of haplotypes analyzed. The 4.5 kbTaq I variant detected by the overlapping cosmid cosOT2 was present in 21 % of haplotypes. Analysis of the RFLP variants with each other revealed seven different haplotypic combinations. Three of the haplotypic combinations were each subdivided into two subsets on the basis of the Nco I RFLP variant they carried at theTNF-B locus. These haplotypic combinations potentially allow differentiation among different extended haplotypes such asHLA-B8, SC01, DR3, HLA-B18, F1 C30, DR3, andHLA-B44, FC31, DR7. The RFLPs detected by the cosmid clones thus provide new tools which will be useful in the further genetic analysis of the MHC class III region.     

A molecular genetic map of the long arm of chromosome 6R of rye incorporating the cereal cyst nematode resistance gene, CreR

 A genetic map of the long arm of chromosome 6R of rye was constructed using eight homoeologous group-6 RFLP clones and five PCR markers derived from the rye-specific dispersed repetitive DNA family, R173. The map was developed using a novel test-cross F₁ (TC-F₁) population segregating for resistance to the cereal cyst nematode. Comparisons were made between the map generated with other rye and wheat group-6 chromosome maps by the inclusion of RFLP clones previously mapped in those species. Co-linearity was observed for common loci. This comparison confirmed a dramatic reduction in recombination for chromosome 6R in the TC-F₁ population. The CreR locus was included in the linkage map via progeny testing of informative TC-F₁ individuals. CreR mapped 3.7 cM distal from the RFLP locus, XksuF37. Comparative mapping should allow the identification of additional RFLP markers more closely linked to the CreR locus. Received: 14 April 1998 / Accepted: 29 April 1998     

Restriction fragment length polymorphism of the major histocompatibility complex of the dog

Human major histocompatibility complex (HLA) cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the DLA-D region in dogs. Genomic DNA from peripheral blood leucocytes of 23 unrelated DLA-D-homozygous dogs representing nine DLA-D types (defined by mixed leucocyte reaction) was digested with restriction enzymes (Bam HI, Eco RI, Hind III, Pvu II, Taq I, Rsa I, Msp I, Pst I, and Bgl II), separated by agarose gel electrophoresis, and transferred onto Biotrace membrane. The Southern blots were successively hybridized with radiolabeled HLA cDNA probes corresponding to DR, DQ, DP, and DO beta genes. The autoradiograms for all nine enzyme digests displayed multiple bands with the DRb, DQb, and DPb probes while the DOb probe hybridized with one to two bands. The RFLP patterns were highly polymorphic but consistent within each DLA-D type. Standard RFLP patterns were established for nine DLA-D types which could be discriminated from each other by using two enzymes (Rsa I and Pst I) and the HLA-DPb probe. Cluster analysis of the polymorphic restriction fragments detected by the DRb probe revealed four closely related supertypic groups or DLA-DR families: Dw3+Dw4+D1, Dw8+D10, D7+D16+D9, and Dw1. This study provides the basis for DLA-D genotyping at a population level by RFLP analysis. These results also suggest that the genetic organization of the DLA-D region may closely resemble that of the HLA complex.     

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).     

Linkage of the Murine Transforming Growth Factor α Gene with Igk, Ly-2, and Fabp1 on Chromosome 6

TGFα is a mitogenic polypeptide found in the conditioned media of transformed cell lines as well as in various solid tumors. Although its physiological role in normal tissues is uncertain, the autocrine action of TGFα on the EGF receptor is postulated to play a role in tumorigenesis. To explore the possibility that pre-existing mouse mutants might have concordance with the mouse TGFα locus (Tgfa) we sought to establish the chromosomal localization of the murine TGFα gene. Using Southern analysis we have detected NcoI and PvuII RFLP in the TGFα gene of progenitor RI mouse strains. These RFLPs have been used to analyze four different RI sets of DNA and to assign Tgfa to the 35-cM region of chromosome 6. Linkage has been established and the data suggest that the distance between Igk and wa-1 anchor loci may be less than 8 cM and that the gene order for the proximal to mid region of mouse chromosome 6 may be: Ggc-Xmmv27-[Brp-1, Lvp-1, Ms6-4]-[Igk, Ly2, Ly3 Odc-rs5, Rn7s-6, Fabp1]-[Tgfa/wa-1]-IL5-R. Homology of synteny has been further defined between the proximal region of mouse chromosome 6 and with the 2p 13-p11 region of human chromosome 2 encompassing TGFA, IGK, CD8A, and FABP1 .     

Restriction fragment length polymorphisms detected by anonymous DNA probes mapped to defined intervals of human chromosome 16

Summary Three anonymous DNA probes ACH207, ACH224, and ACH202, isolated from a flow-purified chromosome 16 library and mapped to defined intervals of human chromosome 16, detected restriction fragment length polymorphisms (RFLPs). The RFLPs were of simple two allele types. The ACH207 (D16S4) probe detected a TaqI and an MspI RFLP with polymorphism information content (PIC) values of 0.30 and 0.27; the ACH224 (D16S5) probe detected and RsaI RFLP, PIC value of 0.34; and the ACH202 (D16S14) probe detected an XbaI RFLP, PIC value of 0.22.     

Integration of the classical and RFLP linkage maps of the short arm of tomato chromosome 1

The classical map of the short arm of chromosome 1 of tomato (Lycopersicon esculentum) has been shown to contain inaccuracies while the RFLP map of this region is known to be generally accurate. Molecular analysis of populations derived from crosses between L. esculentum lines carrying chromosome 1 classical markers and L. pennellii has enabled us to produce an integrated classical and RFLP marker map of this region. New data concerning the linkage relationships between classical markers have also been combined with previous data to produce a new classical map of the short arm of chromosome 1. The orders of the classical markers on these two new maps are in almost complete agreement and are very different to that shown on the previous classical map.     

Mapping the jp (jumbo pollen) gene and QTLs involved in multinucleate microspore formation in diploid alfalfa

The objective of this research was to map the jumbo-pollen trait in diploid alfalfa. Homozygous recessive (jpjp) plants are characterized by the complete failure of post-meiotic cytokinesis during microsporogenesis resulting in 100% 4n-pollen formation. Three F₁ segregating populations were produced and analyzed for pollen-grain production and the segregation of RFLP markers. The first cross combination did not segregate for the jumbo-pollen trait, but showed a clear segregation for multinucleate (bi-, tri- and tetra-nucleate)-microspore formation. Cytological analysis showed that few plants produced 100% normal (uninucleate) microspores, whereas most of them produced multinucleate microspores at a variable frequency (0–75%). Plants with multinucleate microspores always showed a prevalence of binucleated microspores, even though some plants showed a background presence of tri- and tetra-nucleate microspores. QTL analysis based on ANOVA I and Stepwise Multiple Regression identified three QTLs with a highly significant effect on multinucleate-microspore formation. Two cross combinations, subsequently executed, showed Mendelian segregation for the jumbo-pollen trait and were effective in locating the jp gene on linkage group 6 close to the Vg1G1b RFLP locus. Interestingly, this RFLP locus was also linked to one QTL for multinucleate-microspore formation. Genetic models are discussed concerning the presence in linkage group 6 of a cluster of genes involved in multinucleate-microspore formation together with possible relationships between the jp gene and the Vg1G1b QTL. Received: 28 January 1999 / Accepted: 16 December 1999     

The semidwarf gene,sd-1, of rice (Oryza sativa L.). II. Molecular mapping and marker-assisted selection

To establish the location of the semidwarf gene, sd-1, the anthocyanin activator (A), purple node (Pn), purple auricle (Pau), and the isozyme locus, EstI-2, in relation to DNA markers on the molecular linkage map of rice, 20 RFLP markers, previously mapped to the central region of chromosome 1 (McCouch et al. 1988), were mapped onto an F₂ population derived from the cross Taichung 65 (A,Pn,Pau)/Taichung 65 (sd-1). sd-1 and EstI-2 were determined to be linked most tightly to RFLP markers RG 109 and RG 220, which cosegregated with each other. The distance between these RFLP markers and sd-1 was estimated to be 0.8 cM, based on an observed recombination value of 0.8%. The order of genes and markers in this region of chromosome 1 was determined to be sd-1 — (EstI-2 — RG220 — RG109) — RG381 — A — Pn — Pau. To test the efficacy of selection for sd-1 based on these linked markers, 50-day-old F₂ seedlings derived from another cross, Milyang 23/Gihobyeo, were analyzed for marker genotype. At this age, the semidwarf character could not be clearly detected based on phenotype. In addition, plant height was normally distributed in this population, making it difficult to unambiguously identify plants carrying sd-1. Thirteen seedlings homozygous for the sd-1-associated allele at EstI-2, RG220 and RG109, and 13 seedlings homozygous for the Sd-1-associated allele at all three marker loci were selected for further genetic analysis. At 20 days after heading, the culm lengths of these 26 plants were measured and the expected phenotype was confirmed in every case. These 26 plants were then selfed for four generations and F₆ lines were again evaluated to determine whether any recombination among the three molecular markers, or between these markers and the sd-1 gene, could be detected. No recombinants were identified, confirming the tight linkage of these loci and the usefulness of genotypic selection for this recessive semidwarf character prior to the time when it can be evaluated based on phenotype.     

Microcolinearity between a 2-cM region encompassing the grain protein content locus<Emphasis Type="Italic"> Gpc-6B1</Emphasis> on wheat chromosome 6B and a 350-kb region on rice chromosome 2

The conservation of the linear order (colinearity) of genetic markers along large chromosome segments in wheat and rice is well established, but less is known about the microcolinearity between both genomes at subcentimorgan distances. In this study we focused on the microcolinearity between a 2.6-cM interval flanked by markers Xcdo365 and Xucw65 on wheat chromosome 6B and rice chromosome 2. A previous study has shown that this wheat segment includes the Gpc-6B1 locus, which is responsible for large differences in grain protein content (GPC) and is the target of a positional cloning effort in our laboratories. Twenty-one recombination events between Xcdo365 and Xucw65 were found in a large segregating population (935 gametes) and used to map 17 genes selected from rice chromosome 2 in the wheat genetic map. We found a high level of colinearity between a 2.1-cM region flanked by loci Xucw75 and Xucw67 on wheat chromosome 6B and a 350-kb uninterrupted sequenced region in rice chromosome arm 2S. Colinearity between these two genomes was extended to the region proximal to Xucw67 (eight colinear RFLP markers), but was interrupted distal to Xucw75 (six non-colinear RFLP markers). Analysis of different comparative studies between rice and wheat suggests that microcolinearity is more frequently disrupted in the distal region of the wheat chromosomes. Fortunately, the region encompassing the Gpc-6B1 locus showed an excellent conservation between the two genomes, facilitating the saturation of the target region of the wheat genetic map with molecular markers. These markers were used to map the Gpc-6B1 locus into a 0.3-cM interval flanked by PCR markers Xucw79 and Xucw71, and to identify five candidate genes within the colinear 64-kb region in rice.     

High resolution RFLP map around the root knot nematode resistance gene (Mi) in tomato

Summary In the 1940's the root-knot nematode resistance gene (Mi) was introgressed into the cultivated tomato from the wild species, L. peruvianum, and today it provides the only form of genetic resistance against this pathogen. We report here the construction of a high resolution RFLP map around the Mi gene that may aid in the future cloning of this gene via chromosome walking. The map covers the most distal nine map units of chromosome 6 and contains the Mi gene, nine RFLP markers, and one isozyme marker (Aps-1). Based on the analysis of more than 1,000 F₂ plants from four crosses, we were able to pinpoint the Mi gene to the interval between two of these markers — GP79 and Aps-1. In crosses containing the Mi gene, this interval is suppressed in recombination and is estimated to be 0.4 cM in length. In contrast, for a cross not containing Mi, the estimated map distance is approximately 5 times greater (ca. 2 cM).Using RFLP markers around Mi as probes, it was possible to classify nematode resistant tomato varieties into three types based on the amount of linked peruvianum DNA still present. Two of these types (representing the majority of the varieties tested) were found to still contain more than 5 cM of peruvianum chromosome — a result that may explain some of the negative effects (e.g. fruit cracking) associated with nematode resistance. The third type (represented by a single variety) is predicted to carry a very small segment of peruvianum DNA (<2 cM) and may be useful in the identification of additional markers close to Mi and in the orientation of clones during a chromosome walk to clone the gene.     

Assignment of the Ly-6--Ril-1--Sis--H-30--Pol-5/Xmmv-72--Ins-3--Krt-1--Int-1--Gdc-1 region to mouse chromosome 15

Previous work has demonstrated linkage between Ly-6, H-30, and a locus, Ril-1, that affects susceptibility to radiation-induced leukemia. Results of preliminary linkage analyses suggested further that the cluster might be linked to Ly-11 on the proximal portion of mouse chromosome 2. Using molecular probes to examine somatic cell lines and recombinant inbred and congenic strains of mice, we have re-evaluated these linkage relationships. A cloned genomic DNA fragment derived from a retroviral site has been used to define a novel locus, Pol-5, that is tightly linked to both H-30 and Ril-1 as shown by analysis of the B6.C-H-30 ^c congenic mouse strain. Following the segregation of the Pol-5 mouse-specific DNA fragment in a series of somatic cell hybrids carrying various combinations of mouse chromosomes on a rat or Chinese hamster background mapped Pol-5 to mouse chromosome 15. During the course of these studies, restriction fragment length polymorphisms were defined associated with several loci, including Pol-5, Ly-6, Sis, Ins-3, Krt-1, Int-1, and Gdc-1. Three of these loci, Sis, Int-1, and Gdc-1, have been previously mapped to chromosome 15 by others using somatic cell hybrids or isoenzyme analyses. Following the inheritance of these eight loci in recombinant inbred strains of mice allowed the definition of a linkage group on the chromosome with the order Ly-6-Ril-1--Sis--H-30--Pol-5--Ins-3--Krt-1--Int-1--Gdc-1. Analyses of alleles inherited as passengers in B6.C-H-30 ^c, C3H.B-Ly-6 ^b, and C57BL/6By-Eh/+ congenic mouse strains and in situ hybridization experiments support the above gene order and indicate further that the cluster is located on distal chromosome 15, with Ly-6 and Sis near Eh.Abbreviations A agouti - Abl cellular homolog of the Abelson leukemia virus oncogene - Ada adenosine deaminase - Ak-1 adenylate kinase-1 - AXB A/J × C57BL/6J recombinant inbred strain - B2m beta-2 microglobulin - BXA C57BL/6J × A/J recombinant inbred strain - BXD C57BL/6J × DBA/2J recombinant inbred strain - BXH C57BL/6J × C3H/HeJ recombinant inbred strain - CXB BALB/cBy × C57BL/6By recombinant inbred strain - DNA deoxyribonucleic acid - Eh hairy ears - Fpgs folypolyglutamyl synthetase - FXI fractionated x-irradiation - Gdc-1 glycerol phosphate dehydrogenase-1 - Il2r IL-2 receptor - Ins-3 a novel insulinlike gene - Int-1 mammary tumor integration site-1 - Itp inosine triphosphatase - Krt-1 the locus designated here includes a cluster of at least three keratin genes - LTR long terminal repeat - Ly lymphocyte - Lv-6 lymphocyte antigen-6 - Ly-11 lymphocyte antigen-11 - MIH minor histocompatibility - Myc cellular homolog of the Abelson leukemia virus oncogene; pa, pallid; - Pol-5 locus encoding retroviral polymerase-5 - RFLP restriction fragment length polymorphism - RI recombinant inbred mouse strains - Ril-1 radiation-induced leukemia susceptibility-1 locus - SDP strain distribution pattern - Sis cellular homolog of the simian sarcoma virus oncogene - SFFV spleen focus-forming virus - Tpi-1 triosephosphate isomerase-1 - Ve velvet     

RFLP mapping of the vernalization (Vrn1) and frost resistance (Fr1) genes on chromosome 5A of wheat

A population of single chromosome recombinant lines was developed from the cross between a frost-sensitive, vernalization-insensitive substitution line, ‘Chinese Spring’ (Triticum spelta 5A) and a frost-tolerant, vernalization-sensitive line, ‘Chinese Spring’ (‘Cheyenne’ 5A), and used to map the genes Vrn1 and Fr1 controlling vernalization requirement and frost tolerance, respectively, relative to RFLP markers located on this chromosome. The Vrn1 and Fr1 loci were located closely linked on the distal portion of the long arm of 5AL, but contrary to previous observations, recombination between them was found. Three RFLP markers, Xpsr426, Xcdo504 and Xwg644 were tightly linked to both. The location of Vrn1 suggests that it is homoeologous to other spring habit genes in related species, particularly the Sh2 locus on chromosome 7 (5H) of barley and the Sp1 locus on chromosome 5R of rye.     

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).