Fine mapping dissects pleiotropic growth quantitative trait locus into linked loci

A recurring issue in studies of quantitative trait loci (QTLs) is whether QTLs that appear to have pleiotropic effects are indeed caused by pleiotropy at single loci or by linked QTLs. Previous work identified a QTL that affected tail length in mice and the lengths of various bones, including the humerus, ulna, femur, tibia, and mandible. The effect of this QTL on tail length has since been found to be due to multiple linked QTLs and so its apparently pleiotropic effects may have been due to linked QTLs with distinct effects. In the present study we examined a line of mice segregating only for a 0.94-Mb chromosomal region known to contain a subset of the QTLs influencing tail length. We measured a number of skeletal dimensions, including the lengths of the skull, mandible, humerus, ulna, femur, tibia, calcaneus, metatarsus, and a tail bone. The QTL region was found to have effects on the size of the mandible and length of the tail bone, with little or no effect on the other traits. Using a randomization approach, we rejected the null hypothesis that the QTL affected all traits equally, thereby demonstrating that the pleiotropic effects reported earlier were due to linked loci with distinct effects. This result underlines the possibility that seemingly pleiotropic effects of QTLs may frequently be due to linked loci and that high-resolution mapping will often be required to distinguish between pleiotropy and linkage.     

Confirmation of the mapping of the Camurati-Englemann locus to 19q13. 2 and refinement to a 3.2-cM region

Camurati-Englemann syndrome (DPD1) is an autosomal dominant condition associated with progressive cortical sclerosis of the diaphyses of all the long bones. Clinical features include abnormal gait, muscle weakness and wasting, and generalized fatigue. The DPD1 gene was recently mapped to a 15.1-cM region on chromosome 19q13.2. We have narrowed the region containing the DPD1 gene to a 3.2-cM region flanked by short tandem repeat markers, D19S881 and D19S718. TGFB1, a candidate gene mapped within this region, was excluded.     

Fine mapping of the qCTS12 locus, a major QTL for seedling cold tolerance in rice

The temperate japonica rice cultivar M202 is the predominant variety grown in California due to its tolerance to low temperature stress, good grain quality and high yield. Earlier analysis of a recombinant inbred line mapping population derived from a cross between M202 and IR50, an indica cultivar that is highly sensitive to cold stress, resulted in the identification of a number of QTL conferring tolerance to cold-induced wilting and necrosis. A major QTL, qCTS12, located on the short arm of chromosome 12, contributes over 40% of the phenotypic variance. To identify the gene(s) underlying qCTS12, we have undertaken the fine mapping of this locus. Saturating the short arm of chromosome 12 with microsatellite markers revealed that qCTS12 is closest to RM7003. Using RM5746 and RM3103, which are immediately outside of RM7003, we screened 1,954 F₅-F₁₀ lines to find recombinants in the qCTS12 region. Additional microsatellite markers were identified from publicly available genomic sequence and used to fine map qCTS12 to a region of approximately 87 kb located on the BAC clone OSJNBb0071I17. This region contains ten open reading frames (ORFs) consisting of five hypothetical and expressed proteins of unknown function, a transposon protein, a putative NBS-LRR disease resistance protein, two zeta class glutathione S-transferases (OsGSTZ1 and OsGSTZ2), and a DAHP synthetase. Further fine mapping with markers developed from the ORFs delimited the QTL to a region of about 55 kb. The most likely candidates for the gene(s) underlying qCTS12 are OsGSTZ1 and OsGSTZ2.The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Fine mapping of the polled locus to a 1-Mb region on bovine Chromosome 1q12

The absence of horns in Bos taurus is under genetic control of the autosomal dominant polled locus which has been genetically mapped to the centromeric region of cattle Chromosome 1. Recently a 4-Mb BAC contig of this chromosomal region has been constructed. Toward positional cloning of the bovine polled locus, we identified 20 additional microsatellite markers spread over the contig map by random sequencing of bacterial artificial chromosome (BAC) subclones. A total of 26 markers were genotyped in 30 two-generation half-sib families of six different German cattle breeds segregating for the hornless phenotype including 336 informative meioses for the polled character. Our fine-mapping study involving 19 recombinant haplotypes allowed us to narrow the critical region for the bovine polled locus to a 1-Mb segment with a centromeric boundary at RP42-218J17_MS1 and a telomeric boundary at BM6438. For marker-assisted selection purposes, the first evidence of informative flanking markers helps to predict polled genotypes with a higher degree of accuracy within families until testing of the causative mutation is available.     

Mapping a blood pressure quantitative trait locus to a 5.7-cM region in Dahl salt-sensitive rats

A region on rat Chromosome (Chr) 2 of the Dahl salt-sensitive rat (S) was shown previously to contain a quantitative trait locus (QTL) for blood pressure (BP). This was achieved first by linkage, followed by the use of congenic strains. A congenic strain, designated S.MNS-D2Mit6/Adh, contained a segment of Chr 2 from the Milan Normotensive (MNS) rat in the S genetic background. Since the region containing the QTL was roughly 80 cM in size, a further reduction was needed towards the positional or candidate gene cloning. Currently, two congenic substrains were made from the original strain S.MNS-D2Mit6/Adh. One of these two substrains showed a BP-lowering effect, whereas the other substrain did not. Deducing the segment not shared in the two substrains, the BP QTL has to be present in a chromosome region of roughly 5.7 cM between the marker D2Rat303 and the locus for the neutroendopeptidase gene (Nep). Nep is not included within the segment. This region does not seem to contain any candidate genes well known for the BP control. Thus, the final identification of the QTL will most likely lead to the discovery of a brand new gene for the BP regulation. Received: 14 December 2000 / Accepted: 18 January 2001     

Fine mapping a self-fertility locus in perennial ryegrass

Key message

A self-fertility locus was fine mapped to a 1.6 cM region on linkage group 5 in a perennial ryegrass population. This locus was the main determinant of pollen self-compatibility.

Abstract

In grasses, self-incompatibility (SI) is characterized by a two-loci gametophytic (S and Z) mechanism acting together in the recognition and inhibition of self-pollen. Mutations affecting the expression of SI have been reported in a few grass species. In perennial ryegrass (Lolium perenne L.), a mutation independent from S and Z, and mapping on linkage group 5 (LG 5), was previously reported to produce self-fertile plants. Here, we describe fine mapping of the self-fertility (SF) gene in a perennial ryegrass population and determine whether there is any effect of other genomic regions on the pollen compatibility. The phenotypic segregation of SF showed a bimodal distribution with one mean at 49% pollen compatibility and the other at 91%. Marker-trait association analysis showed that only markers on LG 5 were significantly associated with the trait. A single gene model explained 82% of the observed variability and no effects of the other regions were detected. Using segregation and linkage analysis, the SF locus was located to a 1.6 cM region on LG 5. The flanking marker sequences were aligned to rice and Brachypodium distachyon reference genomes to estimate the physical distance. We provide markers tightly linked to SF that can be used for introgression of this trait into advanced breeding germplasm. Moreover, our results represent a further step towards the identification of the SF gene in LG 5.

     

Fine mapping a QTL for carbon isotope composition in tomato

Carbon isotope composition (delta(13)C) and leaf water-use efficiency vary in concert in C3 plants, making delta(13)C useful as a proxy for plant water-use efficiency. A QTL for delta(13)C was detected in the Solanum pennellii chromosome fragment of IL5-4, an introgression line with S. lycopersicum cv. M82 background. M82 and IL 5-4 were crossed, and RFLP markers in the target region converted to PCR-based markers. Forty-one recombinants with an introgression fragment ranging in length from 1.1 to 11.4 cM were identified by marker assisted selection (MAS) among approximately 2000 F2 plants. A total of 29 markers were mapped within the introgression fragment unique to IL5-4. These markers divided the about 9 cM target region into nine intervals. A dominant QTL for delta(13)C, designated QWUE5.1 that explained 25.6% of the total phenotypic variance was mapped to an interval about 2.2 cM long. Twenty-one plants with a S. pennellii chromosome fragment shortened to a length of 2.0-9.1 cM by a second recombination event were generated by MAS of 1,125 F4 plants. Two near isogenic lines with high delta(13)C (small negative value) and carrying QWUE5.1 on the shortest introgression fragments (about 7.0 cM) were identified. The markers and genetic stocks developed are valuable for cloning the gene underlying QWUE5.1, MAS of QWUE5.1, and fine-mapping genes/QTL located in this region.     

Limitations of a two-step moment method for mapping linked multiple QTL

As an alternative to multiple-interval mapping a two-step moment method was recently proposed to map linked multiple quantitative trait loci (QTLs). The advantage of this moment method was supposed to be its simplicity and computational efficiency, especially in detecting closely linked QTLs within a marker bracket, but also in mapping QTLs in different marker intervals. Using simulations it is shown that the two-step moment method may give poor results compared with multiple-interval mapping, irrespective of whether the QTLs are in the same or in different marker intervals, especially if linked QTLs are in repulsion. The criteria of comparison are number of identified QTLs, likelihood ratio test statistics, means and empirical standard errors of the QTL position and QTL effects estimates, and the accuracy of the residual variance estimates. Further, the joint conditional probabilities of QTL genotypes for two putative QTLs within a marker interval were derived and compared with the unmodified approach ignoring the non-independence of the conditional probabilities.     

Detection of novel SNPs and mapping of the fatness QTL on pig chromosome 7q1.1-1.4 region

Many QTLs for fatness traits have been mapped on pig chromosome 7q1.1-1.4 in various pig resource populations. Eight novel markers, including seven SNPs and one insertion or deletion within BTNL1, COL21A1, PPARD, GLP1R, MDFI, GNMT, ABCC10, and PLA2G7 genes, as well as two previously reported SNPs in SLC39A7 and HMGA1 genes, were genotyped in Large White and Meishan pig breeds. Except for two SNPs in HMGA1 and ABCC10 genes, allele frequencies of the other eight markers are highly significant different between Chinese indigenous Meishan breeds and Large White pig breeds. Eight polymorphic sites were then used for linkage and QTL mapping to refine the fatness QTL in a Large White × Meishan F(2) resource population. Five chromosome-wise significant QTLs were detected, of which the QTLs for leaf fat weight, backfat thickness at 6-7th rib and rump, and mean backfat thickness were narrowed to the interval between PPARD and GLP1R genes and the QTL for backfat thickness at thorax-waist between GNMT and PLA2G7 genes on SSC7p1.1-q1.4.     

Fine mapping of a grain-weight quantitative trait locus in the pericentromeric region of rice chromosome 3

As the basis for fine mapping of a grain-weight QTL, gw3.1, a set of near isogenic lines (NILs), was developed from an Oryza sativa, cv. Jefferson x O. rufipogon (IRGC105491) population based on five generations of backcrossing and seven generations of selfing. Despite the use of an interspecific cross for mapping and the pericentromeric location of the QTL, we observed no suppression of recombination and have been able to narrow down the location of the gene underlying this QTL to a 93.8-kb region. The locus was associated with transgressive variation for grain size and grain weight in this population and features prominently in many other inter- and intraspecific crosses of rice. The phenotype was difficult to evaluate due to the large amount of variance in size and weight among grains on a panicle and between grains on primary and secondary panicles, underscoring the value of using multiple approaches to phenotyping, including extreme sampling and NIL group-mean comparisons. The fact that a QTL for kernel size has also been identified in a homeologous region of maize chromosome 1 suggests that this locus, in which the dominant O. rufipogon allele confers small seed size, may be associated with domestication in cereals.     

Use of introgression lines and zonal mapping to identify RAPD markers linked to QTL

RAPD primers were identified as giving parent-specific bands when screened with a set of introgression lines containing introgressed regions of Lycopersicon pennellii that encompass 5 quantitative trait loci affiliated with the production and composition of acylsugars, compounds associated with insect resistance. Primers giving L. pennellii introgression specific bands were zonally mapped to identify bands affiliated with the quantitative trait target and flanking regions using subsets of 7 to 16 F2 individuals which contained small overlapping segments (zones) of the L. pennellii genome spanning those regions. Seventeen RAPD primers, agt-related primers, and an agt clone were then used in mapping the complete F2 population of 144 individuals. This work resulted in the identification of RAPD markers for three of the 5 quantitative trait loci and the construction of an integrated RAPD/RFLP genomic map for tomato (Lycopersicon esculentum x L. pennellii LA716) of 111 RAPD and 8 acylglucose transferase related markers added to a framework map of 150 RFLP markers.     

A comparison of regression interval mapping and multiple interval mapping for linked QTL

Regression interval mapping and multiple interval mapping are compared with regard to mapping linked quantitative trait loci (QTL) in inbred-line cross experiments. For that purpose, a simulation study was performed using genetic models with two linked QTL. Data were simulated for F(2) populations of different sizes and with all QTL and marker alleles fixed for alternative alleles in the parental lines. The criteria for comparison are power of QTL identification and the accuracy of the QTL position and effect estimates. Further, the estimates of the relative QTL variance are assessed. There are distinct differences in the QTL position estimates between the two methods. Multiple interval mapping tends to be more powerful as compared to regression interval mapping. Multiple interval mapping further leads to more accurate QTL position and QTL effect estimates. The superiority increased with wider marker intervals and larger population sizes. If QTL are in repulsion, the differences between the two methods are very pronounced. For both methods, the reduction of the marker interval size from 10 to 5 cM increases power and greatly improves QTL parameter estimates. This contrasts with findings in the literature for single QTL scenarios, where a marker density of 10 cM is generally considered as sufficient. The use of standard (asymptotic) statistical theory for the computation of the standard errors of the QTL position and effect estimates proves to give much too optimistic standard errors for regression interval mapping as well as for multiple interval mapping.     

Fine mapping of a distal chromosome 4 QTL affecting growth and muscle mass in a chicken advanced intercross line

In our previous research, QTL analysis in an F₂ cross between the inbred New Hampshire (NHI) and White Leghorn (WL77) lines revealed a growth QTL in the distal part of chromosome 4. To physically reduce the chromosomal interval and the number of potential candidate genes, we performed fine mapping using individuals of generations F₁₀, F₁₁ and F₁₂ in an advanced intercross line that had been established from the initial F₂ mapping population. Using nine single nucleotide polymorphism (SNP) markers within the QTL region for an association analysis with several growth traits from hatch to 20 weeks and body composition traits at 20 weeks, we could reduce the confidence interval from 26.9 to 3.4 Mb. Within the fine mapped region, markers rs14490774, rs314961352 and rs318175270 were in full linkage disequilibrium (D′ = 1.0) and showed the strongest effect on growth and muscle mass (LOD ≥ 4.00). This reduced region contains 30 genes, compared to 292 genes in the original region. Chicken 60 K and 600 K SNP chips combined with DNA sequencing of the parental lines were used to call mutations in the reduced region. In the narrowed‐down region, 489 sequence variants were detected between NHI and WL77. The most deleterious variants are a missense variant in ADGRA3 (SIFT = 0.02) and a frameshift deletion in the functional unknown gene ENSGALG00000014401 in NHI chicken. In addition, five synonymous variants were discovered in genes PPARGC1A, ADGRA3, PACRGL, SLIT2 and FAM184B. In our study, the confidence interval and the number of potential genes could be reduced 8‐ and 10‐ fold respectively. Further research will focus on functional effects of mutant genes.     

Fine scale mapping of a genetic locus for conditioned fear

Abstract Fear conditioning is one of a number of models for investigating the genetic basis of individual variation in emotion and learning. Genetic mapping using crosses between strains of laboratory mice has identified a locus on chromosome one that appears to influence not only variation in conditioned fear, but also in other validated tests of fear-related behaviour, (including the open-field and the elevated-plus maze), suggesting that the rodent locus may act in ways consistent with how a locus influencing susceptibility to anxiety in humans is believed to operate. Here we use high-resolution mapping in genetically heterogeneous mice to show that a quantitative trait locus influencing conditioned fear can be separated from loci influencing open-field activity. Mapping in two different heterogeneous stocks, the Boulder and Northport HS, gave similar map locations for open-field activity at two positions on the current mouse physical map, one at 162 Mb on chromosome one (negative log P-value 5.4) the other at 173 Mb (negative log P-value 4.8), while mapping of contextual conditioned fear in the Boulder HS identified a locus at 170 Mb (negative log P-value 5.4). Estimates of the 95% confidence intervals show that the locations do not overlap. The region containing a gene or genes that influence variation in conditioned fear is approximately 1 megabase in size and contains only one gene of known function, a pre-B cell leukaemia factor.     

Fine mapping a domestication-related QTL for spike-related traits in a synthetic wheat

QTL analysis using a BC5F2:3 mapping population derived from a cross between Am3, a synthetic hexaploid wheat as a donor parent, and Laizhou953, a Chinese winter wheat cultivar as a recurrent parent, showed that variation at the microsatellite locus Xgwm113 on chromosome 4B was associated with variation in grain number per spike (GN), spike length (SL), and spikelet number per spike (SPI). The Qgn.caas-4B, Qsl.caas-4B, and Qspi.caas-4B were responsible for 16.6%-35.6%, 18.0%-32.3%, and 23.7%-25.9% of the phenotypic variation present in two environments, respectively. Segregation for GN fit a Mendelian monogenic ratio. A subpopulation consisting of 497 plants was used to map the QTL to a 1.2?cM interval between Xgwm113 and Xgwm857. The three spike traits, GN, SL, and SPI, were correlated and were thus probably under the pleiotropic control of the QTL. The Am3 allele had a reduction effect on all three spike traits. Evidence for positive selective history on SSR locus Xgwm113 was supported using Ewens-Watterson's statistic test on a germplasm panel of wild and landrace entries, suggesting that this genomic region may contain genes under selection during wheat domestication.     

Fine mapping of the McLeod locus (XK) to a 150-380-kb region in Xp21.

McLeod syndrome, characterized by acanthocytosis and the absence of a red-blood-cell Kell antigen (Kx), is a multisystem disorder involving a late-onset myopathy, splenomegaly, and neurological defects. The locus for this syndrome has been mapped, by deletion analysis, to a region between the loci for Duchenne muscular dystrophy (DMD) and chronic granulomatous disease (CGD). In this study, we describe a new marker, 3BH/R 0.3 (DXS 709), isolated by cloning the deletion breakpoint of a DMD patient. A long-range restriction map of Xp21, encompassing the gene loci for McLeod and CGD, was constructed, and multiple CpG islands were found clustered in a 700-kb region. Using the new marker, we have limited the McLeod syndrome critical region to 150-380-kb. Within this interval, two CpG-rich islands which may represent candidate sites for the McLeod gene were identified.     

Fine mapping of the Huntington disease linked D4S10 locus by non-radioactive in situ hybridization

Summary The chromosomal localization of a unique DNA fragment, closely linked to Hintington disease (HD), was assessed in situ by hybridization with 2-acetylaminofluorene (AAF) modified probes. In these experiments, a cosmid cloned genomic fragment (c5.5) was used for hybridization. Here we present evidence that confirms the mapping of the D4S10 locus to the p16 region of chromosome 4 and assigns it to the telomere of the short arm.