A YAC contig encompassing the recessive Stargardt disease gene (STGD) on chromosome 1p.

Stargardt disease (STGD) and fundus flavimaculatus are infrequent autosomal recessive conditions characterized by a juvenile macular dystrophy and variable degrees of peripheral retinal changes. Linkage analysis performed in 47 STGD/fundus flavimaculatus families demonstrated significant linkage to 13 polymorphic DNA markers on chromosome 1p. The maximum combined two-point lod score was 32.7 (maximum recombination fraction [phi max] = .006) with the polymorphic marker D1S188. Our data demonstrate that STGD and fundus flavimaculatus are the same disorder clinically and genetically and provide further evidence for genetic homogeneity of this phenotype. Analysis of recombination events on disease chromosomes placed the STGD gene within a 4-cM interval between markers D1S435 and D1S236. A physical map was constructed of a YAC contig flanking STGD, from markers D1S500 to D1S495, and includes the critical interval delineated by historical recombinants. This contig spans approximately 31 cM, with one gap (3-5 cM) that is outside the 4-cM critical region. Localization of STGD to a single YAC contig will facilitate its positional cloning.     

Prospects for association-based fine mapping of a susceptibility gene for a complex disease

The potential of association studies for fine-mapping loci with common disease susceptibility alleles for complex genetic diseases in outbred populations is unclear. For a battery of tightly linked anonymous genetic markers spanning a candidate region centered around a disease locus, simulation methods based on a coalescent process with mutation, recombination, and genetic drift were used to study the spatial distribution of markers with large noncentrality parameters in a case-control study design. Simulations with a disease allele at intermediate frequency, presumably representing an old mutation, tend to exhibit the largest noncentrality parameter values at markers near the disease locus. In contrast, simulations with a disease allele at low frequency, presumably representing a young mutation, often exhibit the largest noncentrality parameter values at markers scattered over the candidate region. In the former case, sample sizes or marker densities sufficient to detect association are likely to lead to useful localization, whereas, in the latter case, localization of the disease locus within the candidate region is much less likely, regardless of the sample size or density of the map. The simulations suggest that for a single marker analysis, the simple strategy of choosing the marker with smallest associated P value to begin a laboratory search for the disease locus performs adequately for a common disease allele.     

Genetic and physical mapping of xa13, a recessive bacterial blight resistance gene in rice

 The recessive gene, xa13, confers resistance to Philippine race 6 (PXO99) of the bacterial blight pathogen Xanthomonas oryzae pv oryzae. Fine genetic mapping and physical mapping were conducted as initial steps in an effort to isolate the gene. Using nine selected DNA markers and two F₂ populations of 132 and 230 plants, xa13 was fine-mapped to a genomic region <4 cM on the long arm of rice chromosome 8, flanked by two RFLP markers, RG136 and R2027. Four DNA markers, RG136, R2027, S14003, and G1149, in the target region were used to identify bacterial artificial chromosome (BAC) clones potentially harboring the xa13 locus from a rice BAC library. A total of 11 BACs were identified, forming four separate contigs including a single-clone contig, 29I3, associated with the RG136 STS marker, the S14003 contig consisting of four clones (44F8, 41O2, 12A16, and 12F20), the G1149 contig with two clones, 23D11 and 21H18, and the R2027 contig consisting of four overlapping clones, 42C23, 30B5, 6B7 and 21H14. Genetic mapping indicated that the xa13 locus was contained in the R2027 contig. Chromosomal walking on the R2027 contig resulted in two more clones, 33C7 and 14L3. DNA fingerprinting showed that the six clones of the R2027 contig were overlapping. Clone 44F8 hybridized with a single fragment from the clone 14L3, integrating the R2027 and S14003 contigs into a single contig consisting of ten BAC clones with a total size of approximately 330 kb. The physical presence of the xa13 locus in the contig was determined by mapping the ends of the BAC inserts generated by TAIL-PCR. In an F₂ population of 230 plants, the BAC-end markers 42C23R and 6B7F flanked the xa13 locus. The probes 21H14F and 21H14R derived from BAC clone 21H14 were found to flank xa13 at a distance of 0.5 cM on either side, using a second F₂ population of 132 plants. Thus, genetic mapping indicated that the contig and the 96-kb clone, 21H14, contained the xa13 locus. Received: 15 August 1998 / Accepted: 29 September 1998     

Genetic characterization and fine mapping of the novel Phytophthora resistance gene in a Chinese soybean cultivar

Phytophthora root rot (PRR), caused by Phytophthora sojae Kaufmann & Gerdemann, is one of the most destructive diseases of soybean [Glycine max (L.) Merr.]. Deployment of resistance genes is the most economical and effective way of controlling the disease. The soybean cultivar ‘Yudou 29’ is resistant to many P. sojae isolates in China. The genetic basis of the resistance in ‘Yudou 29’ was elucidated through an inheritance study and molecular mapping. In response to 25 P. sojae isolates, ‘Yudou 29’ displayed a new resistance reaction pattern distinct from those of differentials carrying known Rps genes. A population of 214 F_2:3 families from a cross between ‘Jikedou 2’ (PRR susceptible) and ‘Yudou 29’ was used for Rps gene mapping. The segregation fit a ratio of 1:2:1 for resistance:segregation:susceptibility within this population, indicating that resistance in ‘Yudou 29’ is controlled by a single dominant gene. This gene was temporarily named RpsYD29 and mapped on soybean chromosome 03 (molecular linkage group N; MLG N) flanked by SSR markers SattWM82-50 and Satt1k4b at a genetic distance of 0.5 and 0.2 cM, respectively. Two nucleotide binding site-leucine rich repeat (NBS-LRR) type genes were detected in the 204.8 kb region between SattWM82-50 and Satt1k4b. These two genes showed high similarity to Rps1k in amino acid sequence and could be candidate genes for PRR resistance. Based on the phenotype reactions and the physical position on soybean chromosome 03, RpsYD29 might be a novel allele at, or a novel gene tightly linked to, the Rps1 locus.     

Genetic analysis and fine mapping of the Ga1-S gene region conferring cross-incompatibility in maize

Cross-incompatibility genes known as gametophyte factors (ga) are numerous in maize. Many popcorn strains carry these genes and cannot be fertilized by pollen of dent and flint maize strains although the reciprocal crosses are successful. A Chinese popcorn strain SDGa25 carries the strongest allele of Ga1 (Ga1-S) and the majority of Chinese dent and flint maize germplasm are incompatible with SDGa25. The incompatibility is due to pollen tube growth obstruction 2 h after pollination. The pollen tube is arrested in the silk segment 5.5 cm distal to the pollination area and never reaches the ovule. The Ga1-S carried by SDGa25 behaves as a single dominant gene. This gene was mapped between markers SD3 on BAC AC200747 0.827 cM apart on the telomere side and SD12 on BAC AC204382 0.709 cM apart on the centromere side. The genetic region mapped spanning the Ga1-S locus was estimated to be 1.5 cM in length and the physical distance is 2,056,343 bp on ctg156 based on the B73 RefGen_v2 sequence. Gametophyte factors influence gene flow direction and the strongest Ga1-S allele is useful for isolating one category of commercial varieties from another. The eight tightly linked markers to Ga1-S developed in this study would greatly improve marker-assisted introgression efficiency and the fine mapping would facilitate the isolation of the Ga1-S.     

Genetic and physical fine mapping of Scmv2, a potyvirus resistance gene in maize

Sugarcane mosaic virus (SCMV) is an important virus pathogen both in European and Chinese maize production, causing serious losses in grain and forage yield in susceptible cultivars. Two major resistance loci confer resistance to SCMV, one located on chromosome 3 (Scmv2) and one on chromosome 6 (Scmv1). We developed a large isogenic mapping population segregating in the Scmv2, but not the Scmv1 region, to minimize genetic variation potentially affecting expression of SCMV resistance. We fine mapped Scmv2 to a region of 0.28 cM, covering a physical distance of 1.3426 Mb, and developed six new polymorphic SSR markers based on publicly available BAC sequences within this region. At present, we still have three recombinants left between Scmv2 and the nearest polymorphic marker on either side of the Scmv2 locus. The region showed synteny to a 1.6 Mb long sequence on chromosome 12 in rice. Analysis of the public B73 BAC library as well as the syntenic rice region did not reveal any similarity to known resistance genes. However, four new candidate genes with a possible involvement in movement of virus were detected.     

Genetic mapping of the erythropoietin receptor gene

We describe a novel, highly informative (polymorphism information content, PIC, = 0.86) simple sequence repeat polymorphism at the 5 end of the gene encoding the human erythropoietin receptor (EPOR) previously assigned to 19pl3.2 by in situ hybridization. Fourteen different allelic size variants were identified in 12 families of the CEPH (Centre d'Etude du Polymorphisme Humain) family panel of 40 families. In pairwise linkage 16 of the 65 chromosome 19 markers reported to the CEPH database gave a lod score exceeding 3.0 when tested against EPOR. The most likely location of EPOR within a framework of 10 markers including orientation and information on reported physical assignments was pter-[INSR-D1 9S177-D19S176]-D 19S24-LDLR-EPOR-cen-D-19S7-D19S49-D19S75-D19S47-APOC2-qter, placing EPOR as the most proximal of the tested loci on the short arm. On an 11-point map the position and order for all other loci except INSR were supported by the data with odds exceeding 1,000:1. The polymorphism at the 5 end of EPOR should provide a useful landmark marker for future mapping studies of this region.     

Genetic analysis and fine mapping of a novel semidominant dwarfing gene LB4D in rice

A dwarf mutant, designated LB4D, was obtained among the progeny of backcrosses to a wild rice introgression line. Genetic analysis of LB4D indicated that the dwarf phenotype was controlled by a single semidominant dwarfing gene, which was named LB4D. The mutants were categorized as dn-type dwarf mutants according to the pattern of internode reduction. In addition, gibberellin (GA) response tests showed that LB4D plants were neither deficient nor insensitive to GA. This study found that tiller formation by LB4D plants was decreased by 40% compared with the wild type, in contrast to other dominant dwarf mutants that have been identified, indicating that a different dwarfing mechanism might be involved in the LB4D dominant mutant. The reduction of plant height in F(1) plants ranged from 27.9% to 38.1% in different genetic backgrounds, showing that LB4D exerted a stronger dominant dwarfing effect. Using large F(2) and F(3) populations derived from a cross between heterozygous LB4D and the japonica cultivar Nipponbare, the LB4D gene was localized to a 46 kb region between the markers Indel 4 and Indel G on the short arm of chromosome 11, and four predicted genes were identified as candidates in the target region.     

Prediction of IBD based on population history for fine gene mapping

A novel multiple regression method (RM) is developed to predict identity-by-descent probabilities at a locus L (IBD_L), among individuals without pedigree, given information on surrounding markers and population history. These IBD_L probabilities are a function of the increase in linkage disequilibrium (LD) generated by drift in a homogeneous population over generations. Three parameters are sufficient to describe population history: effective population size (Ne), number of generations since foundation (T), and marker allele frequencies among founders (p). IBD_L are used in a simulation study to map a quantitative trait locus (QTL) via variance component estimation. RM is compared to a coalescent method (CM) in terms of power and robustness of QTL detection. Differences between RM and CM are small but significant. For example, RM is more powerful than CM in dioecious populations, but not in monoecious populations. Moreover, RM is more robust than CM when marker phases are unknown or when there is complete LD among founders or Ne is wrong, and less robust when p is wrong. CM utilises all marker haplotype information, whereas RM utilises information contained in each individual marker and all possible marker pairs but not in higher order interactions. RM consists of a family of models encompassing four different population structures, and two ways of using marker information, which contrasts with the single model that must cater for all possible evolutionary scenarios in CM.     

Molecular mapping of a recessive gene for resistance to stripe rust in barley

Barley stripe rust, caused by Puccinia striiformis f. sp. hordei, is one of the most important barley (Hordeum vulgare) diseases in the United States. The disease is best controlled using resistant cultivars. Barley genotype Grannenlose Zweizeilige (GZ) has a recessive gene (rpsGZ) that is effective against all races of P. striiformis f. sp. hordei identified so far in the USA. To develop a molecular map for mapping the gene, F₈ recombinant inbred lines (RILs) were developed from the Steptoe X GZ cross through single-seed descent. Seedlings of the parents and RILs were evaluated for resistance to races PSH-14 and PSH-54 of P. striiformis f. sp. hordei under controlled greenhouse conditions. Genomic DNA was extracted from the parents and 182 F₈ RILs and used for linkage analysis. The resistance gene analog polymorphism (RGAP) technique was used to identify molecular markers for rpsGZ. A linkage group for the gene was constructed with 12 RGAP markers, of which two markers co-segregated with the resistance locus, and two markers were closely linked to the locus with a genetic distance of 0.9 and 2.0 cM, respectively. These four markers were present only in the susceptible parent. The closest marker to the resistance allele was 11.7 cM away. Analyses of two sets of barley chromosome addition lines of wheat with the two RGAP markers that were cosegregating with the susceptibility allele showed that rpsGZ and the markers were located on the long arm of barley chromosome 4H. Further, tests with four simple sequence repeat (SSR) markers confirmed the chromosomal location of the rpsGZ gene and also integrated the RGAP markers into the known SSR-based linkage map of barley. The closest SSR marker EBmac0679 had a genetic distance of 7.5 cM with the gene in the integrated linkage map constructed with the 12 RGAP markers and 4 SSR markers. The information on chromosomal location and molecular markers for rpsGZ should be useful for incorporating this gene into commercial cultivars and combining it with other resistance genes for durable resistance.     

Genetic mapping of a dominant gene conferring resistance to cassava mosaic disease

Cassava mosaic disease (CMD) is the most-important disease of cassava (Manihot esculenta) in Africa, and is a potential threat to Latin American (LA) cassava production. Although this viral disease is still unknown in LA, its vector - the whitefly - has recently been found. The disease is best controlled through host-plant resistance, which was first found in third backcross derivatives of an interspecific cross between cassava and Manihot glaziovii, and is thought to be polygenic. Recently, high levels of resistance were also found in several Nigerian cassava landraces. Classical genetic analysis and molecular genetic-mapping of the landraces showed that a major dominant gene confers this resistance. Bulk segregant analysis (BSA) was used to quickly identify a simple sequence repeat (SSR) marker linked to the CMD-resistance gene. The marker, SSRY28, is located on linkage group R of the male-parent-derived molecular genetic map. The gene, designated as CMD2, is flanked by the SSR and RFLP marker GY1 at 9 and 8 cM, respectively. To our knowledge, this is the first report of qualitative virus resistance in cassava, and of molecular markers that tag CMD resistance in cassava. We discuss the use of markers linked to CMD2 for marker-assisted breeding of CMD resistance in Latin America and for increasing the cost-effectiveness of resistance breeding in Africa.     

The ABCR gene in recessive and dominant Stargardt diseases: a genetic pathway in macular degeneration.

Stargardt disease (STGD) is a juvenile-onset macular dystrophy and can be inherited in an autosomal recessive or in an autosomal dominant manner. Genes involved in dominant STDG have been mapped to human chromosomes 13q (STGD2) and 6q (STGD3). Here, we identify a new kindred with dominant STGD and demonstrate genetic linkage to the STGD3 locus. Because of a more severe macular degeneration phenotype of one of the patients in this family, the gene responsible for the recessive STGD1, ABCR, was analyzed for sequence variants in all family members. One allele of the ABCR gene was shown to carry a stop codon-generating mutation (R152X) in three family members, including the one patient who had inherited also the dominant gene. A grandparent of that patient with the same ABCR mutation developed age-related macular degeneration (AMD), consistent with our earlier observation that some variants in the ABCR gene may increase susceptibility to AMD in the heterozygous state. Based on these results, we propose that there is a common genetic pathway in macular degeneration that includes genes for both recessive and dominant STGD.     

Identification, fine mapping and characterization of Rht-dp, a recessive wheat dwarfing (reduced height) gene derived from Triticum polonicum

Semi-dwarfism is an agronomically important trait in breeding for resistance to damage by wind and rain (lodging resistance) and for stable high yields. Dwarf Polish wheat (Triticum polonicum L., 2n = 4x = 28, AABB AS304) is a potential donor of dwarfing and other traits for common wheat improvement. A genetic analysis using an F₂ population derived from a cross of AS304 and tall cultivar AS302 and derived F_2:3 lines indicated that AS304 carries a recessive dwarfing gene, temporarily designated Rht-dp. Molecular markers and bulked segregant analysis were used to characterize and map the gene. Eight polymorphic SSR markers (Xwmc511, Xgwm495, Xgwm 113, Xgwm192, Xgpw7026, Xgpw3017, Xgpw1108 and Xgpw7521) on chromosome arm 4BS and two AFLP markers (M ₈ /E ₅ and M ₄ /E ₃ ) were mapped relative to the dwarfing locus. The closest linked markers, Xgpw3017 and M ₈ /E ₅ at 0.5 and 3.5 cM, respectively, from Rht-dp will enable its marker assisted transfer to wheat breeding populations. Allelic tests indicated that Rht-dp was allelic to Rht-B1b; hence it may be an alternative allele at the Rht-B1 locus.     

Genetic analysis and molecular mapping of a novel recessive gene xa34(t) for resistance against Xanthomonas oryzae pv. oryzae

A new bacterial blight recessive resistance gene xa34(t) was identified from the descendant of somatic hybridization between an aus rice cultivar (cv.) BG1222 and susceptible cv. IR24 against Chinese race V (isolate 5226). The isolate was used to test the resistance or susceptibility of F₁ progenies and reciprocal crosses of the parents. The results showed that F₁ progenies appeared susceptibility there were 128R (resistant):378S (susceptible) and 119R:375S plants in F₂ populations derived from two crosses of BG1222/IR24 and IR24/BG1222, respectively, which both calculates into a 1R:3S ratio. 320 pairs of stochastically selected SSR primers were used for genes?? initial mapping. The screened results showed that two SSR markers, RM493 and RM446, found on rice chromosome 1 linked to xa34(t). Linkage analysis showed that these two markers were on both sides of xa34(t) with the genetic distances 4.29 and 3.05?cM, respectively. The other 50 SSR markers in this region were used for genes?? fine mapping. The further results indicated that xa34(t) was mapped to a 1.42?cM genetic region between RM10927 and RM10591. In order to further narrow down the genomic region of xa34(t), 43 of insertion/deletion (Indel) markers (BGID1-43) were designed according to the sequences comparison between japonica and indica rice. Parents?? polymorphic detection and linkage assay showed that the Indel marker BGID25 came closer to the target gene with a 0.4?cM genetic distance. A contig map corresponding to the locus was constructed based on the reference sequences aligned by the xa34(t) linked markers. Consequently, the locus of xa34(t) was defined to a 204?kb interval flanked by markers RM10929 and BGID25.     

Identification and fine mapping of a mutant gene for palealess spikelet in rice

In grass, the evolutionary relationship between lemma and palea, and their relationship to the flower organs in dicots have been variously interpreted and wildely debated. In the present study, we carried out morphological and genetic analysis of a palealess mutant (pal) from rice (Oryza sativa L.), and fine mapping the gene responsible for the mutated trait. Together, our findings indicate that the palea is replaced by two leaf-like structures in the pal flowers, and this trait is controlled by one recessive gene, termed palealess1 (pal1). With a large F2 segregating population, the pal1 gene was finally mapped into a physical region of 35 kb. Our results also suggest that the lemma and palea of rice are not homologous organs, palea is likely evolutionarily equivalent to the eudicot sepal, and the pal1 should be an A function gene for rice floral organ identity.