Relative fitness of genotypes in a population of Rattus norvegicus polymorphic for warfarin resistance.

Resistance to warfarin and an increased vitamin K requirement appear to be pleiotropic effects of the same allele (Rw2). In a natural population containing resistant individuals where the use of warfarin is discouraged the change in the frequency of resistance should reflect the relative fitnesses of the three possible genotypes. A large polymorphic population of rats was extensively poisoned with warfarin and the level of resistance monitored regularly for a period of 18 months after withdrawal of the poison. During this period the proportion of resistant animals in live-capture samples decreased significantly from approximately 80 per cent to 33 per cent. This decline is consistent with a hypothesis of reduced fitness of both Rw2Rw2 and Rw1Rw2 genotypes relative to Rw1Rw1 under natural conditions. The relative fitnesses of these genotypes were calculated using an optimisation method based on least squares analysis. These estimates were: Rw2Rw2 (0.46), Rw1Rw2 (0.77) and Rw1Rw1 (1.00). Homozygous resistant individuals were found in some of the samples, confirming that the Rw2 allele does not act as a recessive lethal, although it must be extremely disadvantageous. Some heterogeneity was observed in the proportion of resistant animals in samples taken from different areas of the farm building complex. This could reflect stochastic processes influencing the Rw2 allele frequency in small peripheral populations.     

Locus-specific genetic differentiation at Rw among warfarin-resistant rat (Rattus norvegicus) populations

Populations may diverge at fitness-related genes as a result of adaptation to local conditions. The ability to detect this divergence by marker-based genomic scans depends on the relative magnitudes of selection, recombination, and migration. We survey rat (Rattus norvegicus) populations to assess the effect that local selection with anticoagulant rodenticides has had on microsatellite marker variation and differentiation at the warfarin resistance gene (Rw) relative to the effect on the genomic background. Initially, using a small sample of 16 rats, we demonstrate tight linkage of microsatellite D1Rat219 to Rw by association mapping of genotypes expressing an anticoagulant-rodenticide-insensitive vitamin K 2,3-epoxide reductase (VKOR). Then, using allele frequencies at D1Rat219, we show that predicted and observed resistance levels in 27 populations correspond, suggesting intense and recent selection for resistance. A contrast of F(ST) values between D1Rat219 and the genomic background revealed that rodenticide selection has overwhelmed drift-mediated population structure only at Rw. A case-controlled design distinguished these locus-specific effects of selection at Rw from background levels of differentiation more effectively than a population-controlled approach. Our results support the notion that an analysis of locus-specific population genetic structure may assist the discovery and mapping of novel candidate loci that are the object of selection or may provide supporting evidence for previously identified loci.     

Evidence that the separation of Mhc class II from class I loci in the zebrafish,Danio rerio,occurred by translocation

In the zebrafish, Danio rerio, and other teleosts, the class I and class II loci of the major histocompatibility complex ( Mhc) reside on different chromosomes. To shed light on the events that might have generated this difference from tetrapods, in which these two types of loci are clustered in a single chromosomal region, the organization of the class II loci in linkage group 8 of the zebrafish was determined by the characterization of contigs of PAC clones. Three contigs were defined: DAB, DCB, and DBB. The 350-kb-long DAB contig contained only four genes: DDB, DAB, SLC7A4, and DAA. The 150-kb-long DCB contig contained the DCB, DCA, and fz10 genes at an undetermined distance from the DAB contig. And the 120-kb-long DBB contig comprised the DBB gene presumably in another linkage group. The low gene density of the linkage group 8 contigs, contrasting with the high gene density of the zebrafish class I region, and the close association with genes [ SLC7A4 coding for an amino acid transporter, and fz10 (frizzled 10) coding for a receptor of the WNT glycoprotein] that are not linked with the tetrapod Mhc, is interpreted to mean that the separation of the class II from class I loci in teleosts occurred by translocation rather than by genomic or chromosomal duplication.     

Linkage of a gene for DDT-resistance in adults of the mosquito Aedes aegypti.

The gene RDDT2, which gives resistance to DDT in the imago of Aedes aegypti L. has been mapped on linkage group III with respect to six visible markers. The best interpretation of the order of the genes is:- blp - blt - co - fz - wi -RDDT2 - min, but the order blp - blt - co - fz -RDDT2 - wi - min is also possible.     

Sequence, expression, and location of zebrafish frizzled 10

Members of the frizzled gene family encode seven-pass transmembrane proteins that function in the interpretation and reception of Wnt-mediated cell-cell communication events. To investigate frizzled function in early zebrafish development, we isolated the maternally contributed frizzled 10 (fz10) gene and localized it to linkage group 8 using radiation hybrid mapping. The cloned zebrafish fz10 is closely related to the fz10 group from other organisms. Zygotic expression of fz10 is observed in the posterior tail mesenchyme, dorsal neural tube, and different parts of the brain.     

Preliminary investigation of QTLs associated with seedling resistance to ascochyta blight from <Emphasis Type="Italic">Cicer echinospermum</Emphasis>, a wild relative of chickpea

Accessions from Cicer echinospermum, a wild relative of chickpea (Cicer arietinum L.), contain resistance to the fungal disease ascochyta blight, a devastating disease of chickpea. A linkage map was constructed based on an interspecific F(2) population, derived from a cross between a susceptible chickpea cultivar (Lasseter) and a resistant C. echinospermum accession (PI 527930). The linkage map incorporated 83 molecular markers, that included RAPD, ISSR, STMS and RGA markers; eight markers remained unlinked. The map comprised eight linkage groups and covered a map distance of 570 cM. Six out of the eight linkage groups were correlated to linkage groups from the integrated Cicer map using STMS markers. Quantitative trait loci (QTLs) associated with ascochyta blight resistance were detected using interval mapping and single-point analysis. The F(2) population was evaluated for seedling and stem resistance in glasshouse trials. At least two QTLs were identified for seedling resistance, both of which were located within linkage group 4. Five markers were associated with stem resistance, four of which were also associated with seedling resistance. QTLs from previous studies also mapped to LG 4, suggesting that this linkage group is an important region of the Cicer genome for resistance to ascochyta blight.     

RFLP markers for sugar beet breeding: chromosomal linkage maps and location of major genes for rhizomania resistance, monogermy and hypocotyl colour

An RFLP linkage map for the nine chromosomes of sugar beet (Beta vulgaris L. ssp. vulgaris var. altissima Doell) was constructed by using a segregating population from a cross between two plants which were heterozygous for several agronomically interesting characters. One hundred and eleven RFLP loci have been mapped to nine linkage groups using 92 genomic markers. The current RFLP map covers a total length of 540 cM. Evidence for the existence of a major gene for rhizomania resistance (Rr1) is given, together with its map position on linkage group IV in the interval between loci GS44 and GS28a. The presence of an RFLP fragment at the GS3d locus is, until now, the best molecular marker for rhizomania-resistant genotypes in segregating populations of sugar beet; GS3d is linked to Rr1 with 6.7 cM. The gene MM, controlling the polygerm/monogerm seed type, has been mapped on linkage group IX in a distal position at 4.2 cM from the locus GS7. The gene R controlling the hypocotyl colour maps to linkage group VII and does not recombine with the RFLP locus GS42. The inheritance of a group of RFLP loci revealed the possible presence of a translocation in the population used to establish the map. The data presented are discussed in relation to the possibility of using RFLP markers in sugar beet breeding.     

A genetic linkage map of the model legume Lotus japonicus and strategies for fast mapping of new loci

A genetic map for the model legume Lotus japonicus has been developed. The F(2) mapping population was established from an interspecific cross between L. japonicus and L. filicaulis. A high level of DNA polymorphism between these parents was the source of markers for linkage analysis and the map is based on a framework of amplified fragment length polymorphism (AFLP) markers. Additional markers were generated by restriction fragment length polymorphism (RFLP) and sequence-specific PCR. A total of 524 AFLP markers, 3 RAPD markers, 39 gene-specific markers, 33 microsatellite markers, and six recessive symbiotic mutant loci were mapped. This genetic map consists of six linkage groups corresponding to the six chromosomes in L. japonicus. Fluorescent in situ hybridization (FISH) with selected markers aligned the linkage groups to chromosomes as described in the accompanying article by Pedrosa et al. 2002(this issue). The length of the linkage map is 367 cM and the average marker distance is 0.6 cM. Distorted segregation of markers was found in certain sections of the map and linkage group I could be assembled only by combining colormapping and cytogenetics (FISH). A fast method to position genetic loci employing three AFLP primer combinations yielding 89 markers was developed and evaluated by mapping three symbiotic loci, Ljsym1, Ljsym5, and Ljhar1-3.     

Extending the chicken-human comparative map by placing 15 genes on the chicken linkage map

To increase the number of type I loci on the chicken linkage map, chicken genes containing microsatellite sequences (TAn, CAn, GAn, An) were selected from the nucleotide sequence database and primers were developed to amplify the repeats. Initially, 40 different microsatellites located within genes were tested on a panel of animals from diverse breeds, and identified 17 polymorphic microsatellites. These polymorphisms allowed us to add 15 new genes to the chicken linkage map. In addition, two genes were added to the chicken map by fluorescent in situ hybridization. As the map position of the human homologues of 13 of these genes is known, these markers extend the comparative map between chicken and man. Our results confirm and refine conserved regions between chicken and man on chicken chromosomes 2 and 7 and on linkage group E29C09W09. Furthermore, an additional conserved region is identified on chromosome 7.     

Genetic linkage mapping of the soybean aphid resistance gene in PI 243540

The soybean aphid (Aphis glycines Matsumura) is a pest of soybean [Glycine max (L.) Merr.] in many soybean growing countries of the world, mainly in Asia and North America. A single dominant gene in PI 243540 confers resistance to the soybean aphid. The objectives of this study were to identify simple sequence repeat (SSR) markers closely linked to the gene in PI 243540 and to position the gene on the consensus soybean genetic map. One hundred eighty-four F(2) plants and their F(2:3) families from a cross between the susceptible cultivar Wyandot and PI 243540, and the two parental lines were screened with the Ohio biotype of soybean aphid using greenhouse choice tests. A SSR marker from each 10-cM section of the consensus soybean map was selected for bulked segregant analysis (BSA) to identify the tentative genomic location of the gene. The BSA technique was useful to localize the gene to a genomic region in soybean linkage group (LG) F. The entire F(2) population was then screened with polymorphic SSR markers from this genomic region and a linkage map with nine SSR markers flanking the gene was constructed. The aphid resistance gene was positioned in the interval between SSR markers Satt334 and Sct_033 on LG F. These SSR markers will be useful for marker assisted selection of this gene. The aphid resistance gene from PI 243540 mapped to a different linkage group than the only named soybean aphid resistance gene, Rag1, from 'Dowling'. Also, the responses of the two known biotypes of the soybean aphid to the gene from PI 243540 and Rag1 were different. Thus, the aphid resistance gene from PI 243540 was determined to be a new and independent gene that has been named Rag2.     

An apricot (Prunus armeniaca L.) F2 progeny linkage map based on SSR and AFLP markers,mapping plum pox virus resistance and self-incompatibility traits

A genetic linkage map of apricot ( Prunus armeniaca L.) was constructed using AFLP and SSR markers. The map is based on an F(2) population (76 individuals) derived from self-pollination of an F(1) individual ('Lito') originated from a cross between 'Stark Early Orange' and 'Tyrinthos'. This family, designated as 'Lito' x 'Lito', segregated for two important agronomical traits: plum pox virus resistance (PPV) and self-incompatibility. A total of 211 markers (180 AFLPs, 29 SSRs and two agronomic traits) were assigned to 11 linkage groups covering 602 cM of the apricot genome. The average distance (cM/marker) between adjacent markers is 3.84 cM. The PPV resistance trait was mapped on linkage group G1 and the self-incompatibility trait was mapped on linkage group G6. Twenty two loci held in common with other Prunus maps allowed us to compare and establish homologies among the respective linkage groups.     

Genomic assignment of the warfarin resistance locus, Rw, in the rat

The locus responsible for resistance to the anticoagulants warfarin and bromadiolone (locus symbol Rw) was integrated into the rat (Rattus norvegicus) microsatellite genome map. Seventh-generation offspring of a segregating strain of rats heterozygous resistant to both compounds were tested with a blood-clotting-response (BCR) test. No recombination between resistance to warfarin and bromadiolone was observed, indicating a common genetic basis. No recombinants were found between Rw and D1Arb18 (Myl2) located at the MIT-microsatellite map position 95.90 (SHRSP × BN F₂-cross) or 82.24 (FHH × ACI F₂-cross). Resistance segregated in a ratio expected for single, dominant gene responses. An equal number of females and males were resistant, but females retained higher percentage blood coagulation activities (PCA) after anticoagulant administration. Partial synteny between rat, mouse, and human suggests that Myl2 may serve as anchor to map the Rw homologs in mouse and human. Received: 30 December 1998 / Accepted: 17 March 1999     

The Use of Restriction Fragment Length Polymorphisms and DNA Duplications to Study the Organization of the Actin Multigene Family in DICTYOSTELIUM DISCOIDEUM

The techniques of restriction fragment length polymorphism analysis and examination of gene copy number in duplication-bearing Dictyostelium discoideum strains have been used to map four actin genes of the wild-type strain NC4 to specific linkage groups. In part, this was accomplished by identification of restriction fragments corresponding to particular cloned actin genes using gene-specific probes from unique sequence 5' and 3' untranslated regions. Cloned gene Actin 8 (designation act-8) maps to linkage group I; Actins 12 (act-12) and M6 (actM6) to linkage group II. An uncloned gene (act-100) also maps to linkage group II in the same region as actM6, as defined by a chromosomal duplication. From analysis of other wild isolates of D. discoideum, it was determined that in these isolates at least two actin genes map to linkage group I and at least four map to linkage group II. These results demonstrate the utility of molecular techniques in genetic analysis of Dictyostelium, particularly for developmentally regulated genes that have been cloned but that have no identified mutant phenotypes.     

Mapping genes for resistance to Leptosphaeria maculans in Brassica juncea.

Blackleg disease of crucifers, caused by the fungus Leptosphaeria maculans, is a major concern to oilseed rape producers worldwide. Brassica species containing the B genome have high levels of resistance to blackleg. Brassica juncea F2 and first-backcross (B1) populations segregating for resistance to a PG2 isolate of L. maculans were created. Segregation for resistance to L. maculans in these populations suggested that resistance was controlled by two independent genes, one dominant and one recessive in nature. A map of the B. juncea genome was constructed using segregation in the F2 population of a combination of restriction fragment length polymorphism (RFLP) and microsatel lite markers. The B. juncea map consisted of 325 loci and was aligned with previous maps of the Brassica A and B genomes. The gene controlling dominant resistance to L. maculans was positioned on linkage group J13 based on segregation for resistance in the F2 population. This position was confirmed in the B1 population in which the resistance gene was definitively mapped in the interval flanked by pN199RV and sB31143F. The provisional location of the recessive gene controlling resistance to L. maculans on linkage group J18 was identified using a subset of informative F2 individuals.     

Chromosomal mapping of tRNA genes from Dictyostelium discoideum

Summary Different wild-type isolates of Dictyostelium discoideum exhibit extensive polymorphism in the length of restriction fragments carrying tRNA genes. These size differences were used to study the organisation of two tRNA gene families which encode a tRNA^Val(GUU) and a tRNA^Val(GUA) gene. The method used involved a combination of classitics. The tRNA genes were mapped to specific linkage groups (chromosomes) by correlating the presence of polymorphic DNA bands that hybridized with the tRNA gene probes with the presence of genetic markers for those linkage groups. These analyses established that both of the tRNA gene families are dispersed among sites on several of the chromosomes. Information of nine tRNA^Val(GUU) genes from the wild-type isolate NC4 was obtained: three map to linkage group I (C, E, F,), two map to linkage group II (D, I), one maps to linkage group IV (G), one, which corresponds to the cloned gene, maps to either linkage group III or VI (B), and two map to one of linkage groups III, VI or VIII (A, H). Six tRNA^Val(GUA) genes from the NC4 isolate were mapped; one to linkage group I (D), two to linkage group III, VI or VII (B, C) and three to linkage group VII or III (A, E, F).     

A comprehensive linkage map of the pig based on a wild pig - Large White intercross

A comprehensive linkage map, including 236 linked markers with a total sex-average map length of about 2300 cM, covering nearly all parts of the pig genome has been established. Linkage groups were assigned to all 18 autosomes, the X chromosome and the X/Y pseudoautosomal region. Several new gene assignments were made including the assignment of linkage group U1 (EAK-HPX) to chromosome 9. The linkage map includes 77 type I loci informative for comparative mapping and 72 in situ mapped markers physically anchoring the linkage groups on chromosomes. A highly significant heterogeneity in recombination rates between sexes was observed with a general tendency towards an excess of female recombination. The average ratio of female to male recombination was estimated at 1–4:1 but this parameter varied between chromosomes as well as between regions within chromosomes. An intriguing finding was that blood group loci were overrepresented at the distal ends of linkage groups.     

Genetic Analysis of the Reciprocal Translocation T2(I;VIII) of Aspergillus Using the Technique of Mitotic Mapping in Homozygous Translocation Diploids

A UV-induced sulphite-requiring mutant (sD50) consistently shows mitotic linkage to groups I and VIII in haploids from heterozygous mapping diploids. This linkage was found to be due to a reciprocal translocation T2(I;VIII) which could not be separated from the sulphite requirement in about 100 tested progeny from heterozygous crosses, and both may well have been induced by the same mutational event. T2(I;VIII) is the first case of a reciprocal translocation in Aspergillus which showed meiotic linkages between markers of different linkage groups, and, in addition, involved chromosome arms containing markers suitable for complete mapping by the technique of mitotic recombination in homozygous translocation diploids.-Using various selective markers, haploid segregants and diploid crossovers of all possible types were isolated from homozygous translocation diploids. (1) Haploid segregants showed new linkage relationships in T/T diploids: all available markers of VIII now segregated as a group with the majority of the markers of I, except for the markers of the left tip of I. These formed a separate linkage group and are presumably translocated to VIII. (2) Diploid mitotic crossovers confirmed this information and showed that the orientation of the translocated segments was unchanged. These findings conclusively demonstrate that T2(I;VIII) is a reciprocal translocation due to an exchange of the left tip of group I with the long right arm of group VIII.-Since the position of the break on VIIIR was found to be at sD50 this marker could be used to map the break on IL by meiotic recombination in heterozygous crosses. In addition, such crosses showed reduced recombination around the breaks, so that it was possible to sequence markers which normally are barely linked.     

Construction of genetic linkage maps and QTL mapping for X-disease resistance in tetraploid chokecherry (Prunus virginiana L.) using SSR and AFLP markers

Chokecherry (Prunus virginiana L.) (2n = 4x = 32) is a unique Prunus species for both genetics and disease resistance research due to its tetraploid nature and known variations in X-disease resistance. X-disease is a destructive disease of stone fruit trees, causing yield loss and poor fruit quality. However, genetic and genomic information on chokecherry is limited. In this study, simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers were used to construct genetic linkage maps and to identify quantitative trait loci (QTLs) associated with X-disease resistance in chokecherry. A segregating population (101 progenies) was developed by crossing an X-disease-resistant chokecherry line (RC) with a susceptible chokecherry line (SC). A total of 498 DNA markers (257 SSR and 241 AFLP markers) were mapped on the two genetic maps of the two parental lines (RC and SC). The map of RC contains 302 markers assigned to 14 linkage groups covering 2,089 cM of the genome. The map of SC has 259 markers assigned to 16 linkage groups covering 1,562.4 cM of the genome. The average distance between two markers was 6.9 cM for the RC map and 6.0 cM for the SC map. One QTL located on linkage group 15 on the map of SC was found to be associated with X-disease resistance. Genetic linkage maps and the identified QTL linked to X-disease resistance will further facilitate genetic research and breeding of X-disease resistance in chokecherry and other Prunus species.     

An interspecific (Capsicum annuum ×C. chinese) F2 linkage map in pepper using RFLP and AFLP markers

We have constructed a molecular linkage map of pepper (Capsicum spp.) in an interspecific F₂ population of 107 plants with 150 RFLP and 430 AFLP markers. The resulting linkage map consists of 11 large (206–60.3 cM) and 5 small (32.6–10.3 cM) linkage groups covering 1,320 cM with an average map distance between framework markers of 7.5 cM. Most (80%) of the RFLP markers were pepper-derived clones, and these markers were evenly distributed across the genome. By using 30 primer combinations, we were able to generate 444 AFLP markers in the F₂ population. The majority of the AFLP markers clustered in each linkage group, although PstI/MseI markers were more evenly distributed than EcoRI/MseI markers within the linkage groups. Genes for the biosynthesis of carotenoids and capsaicinoids were mapped on our linkage map. This map will provide the basis of studying secondary metabolites in pepper. Received: 20 October 1999 / Accepted: 3 July 2000