Quantitative trait locus analysis using recombinant inbred intercrosses: theoretical and empirical considerations

We describe a new approach, called recombinant inbred intercross (RIX) mapping, that extends the power of recombinant inbred (RI) lines to provide sensitive detection of quantitative trait loci (QTL) responsible for complex genetic and nongenetic interactions. RIXs are generated by producing F1 hybrids between all or a subset of parental RI lines. By dramatically extending the number of unique, reproducible genomes, RIXs share some of the best properties of both the parental RI and F2 mapping panels. These attributes make the RIX method ideally suited for experiments requiring analysis of multiple parameters, under different environmental conditions and/or temporal sampling. However, since any pair of RIX genomes shares either one or no parental RIs, this cross introduces an unusual population structure requiring special computational approaches for analysis. Herein, we propose an efficient statistical procedure for QTL mapping with RIXs and describe a novel empirical permutation procedure to assess genome-wide significance. This procedure will also be applicable to diallel crosses. Extensive simulations using strain distribution patterns from CXB, AXB/BXA, and BXD mouse RI lines show the theoretical power of the RIX approach and the analysis of CXB RIXs demonstrates the limitations of this procedure when using small RI panels.     

Genetic analysis and characterization of a new maize association mapping panel for quantitative trait loci dissection

Association mapping based on the linkage disequilibrium provides a promising tool to identify genes responsible for quantitative variations underlying complex traits. Presented here is a maize association mapping panel consisting of 155 inbred lines with mainly temperate germplasm, which was phenotyped for 34 traits and genotyped using 82 SSRs and 1,536 SNPs. Abundant phenotypic and genetic diversities were observed within the panel based on the phenotypic and genotypic analysis. A model-based analysis using 82 SSRs assigned all inbred lines to two groups with eight subgroups. The relative kinship matrix was calculated using 884 SNPs with minor allele frequency ≥20% indicating that no or weak relationships were identified for most individual pairs. Three traits (total tocopherol content in maize kernel, plant height and kernel length) and 1,414 SNPs with missing data <20% were used to evaluate the performance of four models for association mapping analysis. For all traits, the model controlling relative kinship (K) performed better than the model controlling population structure (Q), and similarly to the model controlling both population structure and relative kinship (Q + K) in this panel. Our results suggest this maize panel can be used for association mapping analysis targeting multiple agronomic and quality traits with optimal association model.     

The F7 gene and clotting factor VII levels: dissection of a human quantitative trait locus

Localization of human quantitative trait loci (QTLs) is now routine. However, identifying their functional DNA variants is still a formidable challenge. We present a complete dissection of a human QTL using novel statistical techniques to infer the most likely functional polymorphisms of a QTL that influence plasma levels of clotting factor VII (FVII), a risk factor for cardiovascular disease. Resequencing of 15 kb in and around the F7 gene identified 49 polymorphisms, which were then genotyped in 398 people. Using a Bayesian quantitative trait nucleotide (BQTN) method, we identified four to seven functional variants that completely account for this QTL. These variants include both rare coding variants and more common, potentially regulatory polymorphisms in intronic and promoter regions.     

Characterization of a major X-linked quantitative trait locus influencing body weight of mice

Growth rate in mice is an archetypal quantitative trait that has long been studied genetically, physiologically, and metabolically, but its genetic basis is still poorly understood due to its complex inheritance and the influence of environment. We measured differences in 17 growth-related traits between a pair of partially congenic lines that differ for a segment of the X chromosome containing a quantitative trait locus (QTL) that we identified in a genomewide QTL scan. The QTL has a large effect on mean body weight of approximately 20% at all ages, and affects early growth rate to a greater extent than late growth rate. Feed is converted to body mass more efficiently in the high chromosome segment-bearing line than the low line. The weights of various internal organs are affected to a somewhat greater extent by the QTL than body weight. The proportional change in body length is smaller than body weight, but this may be an effect of scale. Body weight at late ages appears to allow the most efficient detection of allelic differences at the QTL, although assignment of genotypic state based on phenotype is never unambiguous.     

Genetic mapping of quantitative trait loci affecting body weight, egg character and egg production in F2 intercross chickens

Phenotypic measurements of chicken egg character and production traits are restricted to mature females only. Marker assisted selection of immature chickens using quantitative trait loci (QTL) has the potential to accelerate the genetic improvement of these traits in the chicken population. The QTL for 12 traits (i.e. body weight (BW), six for egg character, three for egg shell colour and two for egg production) of chickens were identified. An F2 population comprising 265 female chickens obtained by crossing White Leghorn and Rhode Island Red breeds and genotyped for 123 microsatellite markers was used for detecting QTL. Ninety-six markers were mapped on 25 autosomal linkage groups, and 13 markers were mapped on one Z chromosomal linkage group. Eight previous unmapped markers were assigned to their respective chromosomes in this study. Significant QTL were detected for BW on chromosomes 4 and 27, egg weight on chromosome 4, the short length of egg on chromosome 4, and redness of egg shell colour (using the L*a*b* colour system) on chromosome 11. A significant QTL on the Z chromosome was linked with age at first egg. Significant QTL could account for 6-19% of the phenotypic variance in the F2 population.     

Genetic analysis of an F2 intercross between two strains of Japanese quail provided evidence for quantitative trait loci affecting carcass composition and internal organs

The purpose of this study was to identify genomic regions, quantitative trait loci (QTL), affecting carcass traits on chromosome 1 in an F2 population of Japanese quail. For this purpose, two white and wild strains of Japanese quail (16 birds) were crossed reciprocally and F1 generation (34 birds) was created. The F2 generation was produced by intercrossing of the F1 birds. Phenotypic data including carcass weight, internal organs and carcass parts were collected on F2 animals (422 birds). The total mapping population (472 birds) was genotyped for 8 microsatellite markers on chromosome 1. QTL analysis was performed with interval mapping method applying the line-cross model. Significant QTL were identified for breast weight at 0 (P < 0.01), 172 (P < 0.05) and 206 (P < 0.01), carcass weight at 91 (P < 0.05), carcass fatness at 0 (P < 0.01), pre-stomach weight at 206 (P < 0.01) and uropygial weight gland at 197 (P < 0.01) cM on chromosome 1. There was also evidence for imprinted QTL affecting breast weight (P < 0.01) on chromosome 1. The proportion of the F2 phenotypic variation explained by the significant additive, dominance and imprinted QTL effects ranged from 1.0 to 7.3 %, 1.2 to 3.3 % and 1.4 to 2.2 %, respectively.     

Genetic dissection of tocopherol and phytosterol in recombinant inbred lines of sunflower through quantitative trait locus analysis and the candidate gene approach

Sunflower (Helianthus annuus L.) contains tocopherol, a non-enzymatic antioxidant known as lipid-soluble vitamin E, and phytosterol, with interesting properties, which can result in decreased risk of chronic diseases in humans and with several beneficial effects in plants. The genetic control of tocopherol and phytosterol content in a population of 123 recombinant inbred lines of sunflower was studied through quantitative trait loci (QTL) analysis using 190 simple sequence repeats and a gene-based linkage map. Seven experiments were conducted in different environments in France and Iran during 2007 and 2008. Each experiment consisted of three replications. Means over all environments were used for QTL mapping. Five QTL for total tocopherol content on linkage groups 1, 8, 10 and 14 accounted for 45% of phenotypic variation, whereas four QTL for total phytosterol content on linkage groups 1, 2, 16 and 17 explained 27% of the phenotypic variation. GST, PAT2, SFH3 and POD genes showed co-localization with QTL for total phytosterol content. SMT2 is also mapped on linkage group 17 near the QTL of total phytosterol content. Four candidate genes, VTE4, HPPD, GST and Droug1, exhibited co-localization with QTL for total tocopherol content. The candidate genes associated with tocopherol and phytosterol, especially HPPD, VTE4 and SMT2, could be used for alternation of the tocopherol and phytosterol content of sunflower seeds through the development of functional markers.     

Diabetic modifier QTLs identified in F2 intercrosses between Akita and A/J mice

To identify novel genetic modifiers of type 2 diabetes (T2D), we performed quantitative trait loci (QTL) analysis on F₂ progeny of hypoinsulinemic diabetic Akita mice, heterozygous for the Ins2 gene Cys96Tyr mutation, and nondiabetic A/J mice. We generated 625 heterozygous (F₂-Hetero) and 338 wild-type (F₂-Wild) mice with regard to the Ins2 mutation in F₂ intercross progeny. We measured quantitative traits, including plasma glucose and insulin concentrations during the intraperitoneal glucose tolerance test (IPGTT), and body weight (BW). We observed three significant QTLs in hypoinsulinemic hyperglycemic male F₂-Hetero mice, designated Dbm1, Dbm3, and Dbm4 on Chromosomes 6, 14, and 15, respectively. They showed linkage to plasma glucose concentrations, with significant maximum logarithm of odds (LOD) scores of 4.12, 4.17, and 6.17, respectively, all exceeding threshold values by permutation tests. In normoinsulinemic normoglycemic male F₂-Wild mice, Dbm1 on Chromosome 6 showed linkage to both plasma insulin concentrations and BW, and Dbm2 on Chromosome 11 showed linkage to plasma glucose concentrations only, with LOD scores of 4.52 and 6.32, and 5.78, respectively. Based on these results, we concluded that Dbm1, Dbm2, Dbm3, and Dbm4 represent four major modifier QTLs specifically affecting T2D-related traits and that these diabetic modifier QTLs are conditional on the heterozygous Ins2 gene mutation and sex to exert their modifier functions. Identification of the genes responsible for these QTLs would provide new drug development targets for human T2D. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Joint mapping of quantitative trait loci using F2 populations

In this paper, the theory of joint mapping of quantitative trait loci is extended to F₂ populations. Two independent regression equations, related to the additive and dominance effects respectively, are derived. Therefore, there are three alternative strategies for mapping QTLs, called additive-based mapping (ABM), dominance-based mapping (DBM) and additive-dominance-based mapping (ADBM). Simulation results have shown that ADBM is the most appropriate in most situations.     

Genetic basis of climatic adaptation in scots pine by bayesian quantitative trait locus analysis

We examined the genetic basis of large adaptive differences in timing of bud set and frost hardiness between natural populations of Scots pine. As a mapping population, we considered an "open-pollinated backcross" progeny by collecting seeds of a single F(1) tree (cross between trees from southern and northern Finland) growing in southern Finland. Due to the special features of the design (no marker information available on grandparents or the father), we applied a Bayesian quantitative trait locus (QTL) mapping method developed previously for outcrossed offspring. We found four potential QTL for timing of bud set and seven for frost hardiness. Bayesian analyses detected more QTL than ANOVA for frost hardiness, but the opposite was true for bud set. These QTL included alleles with rather large effects, and additionally smaller QTL were supported. The largest QTL for bud set date accounted for about a fourth of the mean difference between populations. Thus, natural selection during adaptation has resulted in selection of at least some alleles of rather large effect.     

Genetic dissection of wheat panicle traits using linkage analysis and a genome-wide association study

Key message

Coincident regions on chromosome 4B for GW, on 5A for SD and TSS, and on 3A for SL and GNS were detected through an integration of a linkage analysis and a genome-wide association study (GWAS). In addition, six stable QTL clusters on chromosomes 2D, 3A, 4B, 5A and 6A were identified with high PVE% on a composite map.

Abstract

The panicle traits of wheat, such as grain number per spike and 1000-grain weight, are closely correlated with grain yield. Superior and effective alleles at loci related to panicles developments play a crucial role in the progress of molecular improvement in wheat yield breeding. Here, we revealed several notable allelic variations of seven panicle-related traits through an integration of genome-wide association mapping and a linkage analysis. The linkage analysis was performed using a recombinant inbred line (RIL) population (173 lines of F_8:9) with a high-density genetic map constructed with 90K SNP arrays, Diversity Arrays Technology (DArT) and simple sequence repeat (SSR) markers in five environments. Thirty-five additive quantitative trait loci (QTL) were discovered, including eleven stable QTLs on chromosomes 1A, 2D, 4B, 5B, 6B, and 6D. The marker interval between EX_C101685 and RAC875_C27536 on chromosome 4B exhibited pleiotropic effects for GW, SL, GNS, FSN, SSN, and TSS, with the phenotypic variation explained (PVE) ranging from 5.40 to 37.70%. In addition, an association analysis was conducted using a diverse panel of 205 elite wheat lines with a composite map (24,355 SNPs) based on the Illumina Infinium assay in four environments. A total of 73 significant marker-trait associations (MTAs) were detected for panicle traits, which were distributed across all wheat chromosomes except for 4D, 5D, and 6D. Consensus regions between RAC875_C27536_611 and Tdurum_contig4974_355 on chromosome 4B for GW in multiple environments, between QTSS5A.7-43 and BS00021805_51 on 5A for SD and TSS, and between QSD3A.2-164 and RAC875_c17479_359 on 3A for SL and GNS in multiple environments were detected through linkage analysis and a genome-wide association study (GWAS). In addition, six stable QTL clusters on chromosomes 2D, 3A, 4B, 5A, and 6A were identified with high PVE% on a composite map. This study provides potentially valuable information on the dissection of yield-component traits and valuable genetic alleles for molecular-design breeding or functional gene exploration.

     

Genetic dissection of drought tolerance and recovery potential by quantitative trait locus mapping of a diploid potato population

Potato is the third most important staple food crop in terms of consumption, yet it is relatively susceptible to yield loss because of drought. As a first step towards improving drought tolerance in this crop, we set out to identify the genetic basis for drought tolerance in a diploid potato mapping population. Experiments were carried out under greenhouse conditions in two successive years by recording four physiological, seven growth and three yield parameters under stress and recovery treatments. Genotypes showed significant variation for drought and recovery responses. The traits measured had low to moderately high heritabilities (ranging from 22 to 74?%). A total of 47 quantitative trait loci (QTL) were identified, of which 28 were drought-specific, 17 under recovery treatment and two under well-watered conditions. The majority of these growth and yield QTL co-localized with a QTL for maturity on chromosome 5. Four QTL for ??¹³C, three for chlorophyll content and one for chlorophyll fluorescence (F _v/F _m) were found to co-localize with yield and other growth trait QTL identified on other chromosomes. Several multi-year and multi-treatment QTL were detected and QTL?×?environment interaction was found for ??¹³C. To our knowledge, this is the first comprehensive QTL study on water deficit and recovery potential in potato.     

Genetic dissection of tocopherol content and composition in maize grain using quantitative trait loci analysis and the candidate gene approach

Tocopherols are essential micronutrients for humans and animals, with several beneficial effects in plants. Among cereals, only maize grains contain high concentrations of tocopherols. In this investigation we analyzed, during 2004 and 2005, by high-performance liquid chromatography (HPLC), a population of 233 recombinant inbred lines (RIL) which were derived from two diverse parents and had extremely variable tocopherol content and composition. A genetic map was constructed using 208 polymorphic molecular markers including gene-targeted markers based on six candidate genes of the tocopherol biosynthesis pathway (HPPD, VTE1, VTE3, VTE4, P3VTE5, and P4VTE5). Thirty-one quantitative trait loci (QTL) associated with quantitative variation of tocopherol content and composition were identified by composite interval mapping (CIM); these were located on sixteen genomic regions covering all the chromosomes except chromosome 4. Most (65%) QTL were co-located, suggesting that in some cases the same QTL predominantly affected the amounts of more than one tocopherol. Two candidate genes, HPPD and VTE4 showed co-localization with major QTL for tocopherol content and composition whereas only one interval (umc1075–umc1304) on chromosome eight exhibited a QTL for α, δ, γ, and total tocopherols with high LOD and PVE values. The candidate genes associated with tocopherol content and with composition, especially VTE4 and HPPD, could be precisely used for alteration of the tocopherol content and composition of maize grains by development of functional markers. Other identified major QTL especially those on chromosomes 8, 1, and 2 (near candidate gene VTE5) can also be used for improvement of maize grain quality by marker-assisted selection.     

Quantitative trait locus analysis of longitudinal quantitative trait data in complex pedigrees

There is currently considerable interest in genetic analysis of quantitative traits such as blood pressure and body mass index. Despite the fact that these traits change throughout life they are commonly analyzed only at a single time point. The genetic basis of such traits can be better understood by collecting and effectively analyzing longitudinal data. Analyses of these data are complicated by the need to incorporate information from complex pedigree structures and genetic markers. We propose conducting longitudinal quantitative trait locus (QTL) analyses on such data sets by using a flexible random regression estimation technique. The relationship between genetic effects at different ages is efficiently modeled using covariance functions (CFs). Using simulated data we show that the change in genetic effects over time can be well characterized using CFs and that including parameters to model the change in effect with age can provide substantial increases in power to detect QTL compared with repeated measure or univariate techniques. The asymptotic distributions of the methods used are investigated and methods for overcoming the practical difficulties in fitting CFs are discussed. The CF-based techniques should allow efficient multivariate analyses of many data sets in human and natural population genetics.     

Genetic dissection of collagen-induced arthritis in Chromosome 10 quantitative trait locus speed congenic rats: evidence for more than one regulatory locus and sex influences

Rat Chromosome 10 (RNO10) harbors Cia5, a non-MHC quantitative trait locus (QTL) that regulates the severity of type II collagen-induced arthritis (CIA) in DAxF344 and DAxBN F2 rats. CIA is an animal model with many features that resemble rheumatoid arthritis. To facilitate analysis of Cia5 independently of the other CIA regulatory loci on other chromosomes, DA recombinant QTL speed congenic rats, DA.F344(Cia5), were generated. These QTL congenic rats have a large chromosomal segment containing Cia5 (interval size < or =80.1 cM) from CIA-resistant F344 rats introgressed into their genome. Phenotypic analyses of these rats for susceptibility and severity of CIA confirmed that Cia5 is an important disease-modifying locus. CIA severity was significantly lower in the Cia5 congenic rats than in DA controls. We also generated DA Cia5 speed sub-congenic rats, DA.F344(Cia5a), which had a smaller segment of the F344 genome, Cia5a, comprising only the distal q-telomeric end (interval size < or = 22.5 cM) of Cia5, introgressed into their genome. DA.F344(Cia5a) sub-congenic rats also exhibited reduced CIA disease severity compared with the parental DA rats. The regulatory effects in both congenic strains were sex influenced. The disease-ameliorating effect of the larger fragment, Cia5, was greater in males than in females, but the effect of the smaller fragment, Cia5a, was greater in females. We also present an improved genetic linkage map covering the Cia5/Cia5a region, which we have integrated with two rat radiation hybrid maps. Comparative homology analysis of this genomic region with mouse and human chromosomes was also undertaken. Regulatory loci for multiple autoimmune/inflammatory diseases in rats (RNO10), mice (MMU11), and humans (HSA17 and HSA5q23-q31) map to chromosomal segments homologous to Cia5 and Cia5a.     

Quantitative trait locus analysis of leaf dissection in tomato using Lycopersicon pennellii segmental introgression lines

Leaves are one of the most conspicuous and important organs of all seed plants. A fundamental source of morphological diversity in leaves is the degree to which the leaf is dissected by lobes and leaflets. We used publicly available segmental introgression lines to describe the quantitative trait loci (QTL) controlling the difference in leaf dissection seen between two tomato species, Lycopersicon esculentum and L. pennellii. We define eight morphological characteristics that comprise the mature tomato leaf and describe loci that affect each of these characters. We found 30 QTL that contribute one or more of these characters. Of these 30 QTL, 22 primarily affect leaf dissection and 8 primarily affect leaf size. On the basis of which characters are affected, four classes of loci emerge that affect leaf dissection. The majority of the QTL produce phenotypes intermediate to the two parent lines, while 5 QTL result in transgression with drastically increased dissection relative to both parent lines.     

Congenic strains reveal effects of the epilepsy quantitative trait locus,El2, separate from other El loci

Congenic mouse strains made by transferring epilepsy predisposing alleles El1, El2, and El3 from the EL/Suz strain to the ABP/Le recipient were tested for seizure frequency following gentle rhythmic stimulation. Mice homozygous for El2, but not El1 or El3, experienced seizures much more frequently than ABP controls, while respective El1 homozygotes and El2 heterozygotes had only a modest increase over ABP, and El3 homozygotes showed no increase. Association between marker genotypes and seizure trequency in small intra-strain crosses showed that the phenotypic effects of El2 map to the selected interval, and that segregation of El2 accounts for virtually all genetic effects. However, in separating El2 from other EL susceptibility alleles, the seizure frequency phenotype was weaker and less heritable than in crosses between parental strains. These results confirm El2 as an important QTL and show that it has significant phenotypic effects in the absence of other EL-derived alleles, including El1. In addition, the present localization of El2 on Chr 2 suggests several potential candidate genes for El2, including the subunit of phospholipase-C. The approach to dissecting complex traits by making congenic strains for individual QTL is discussed.Center for Medical Genetics, Marshfield Medical Research Foundation, Marshfield, Wisconsin, 54449, USA     

Genetic variation in the pleiotropic association between physical activity and body weight in mice

Background

A sedentary lifestyle is often assumed to lead to increases in body weight and potentially obesity and related diseases but in fact little is known about the genetic association between physical activity and body weight. We tested for such an association between body weight and the distance, duration, and speed voluntarily run by 310 mice from the F₂ generation produced from an intercross of two inbred lines that differed dramatically in their physical activity levels.

Methods

We used a conventional interval mapping approach with SNP markers to search for QTLs that affected both body weight and activity traits. We also conducted a genome scan to search for relationship QTLs (relQTLs), or chromosomal regions that affected an activity trait variably depending on the phenotypic value of body weight.

Results

We uncovered seven quantitative trait loci (QTLs) affecting body weight, but only one co-localized with another QTL previously found for activity traits. We discovered 19 relQTLs that provided evidence for a genetic (pleiotropic) association of physical activity and body weight. The three genotypes at each of these loci typically exhibited a combination of negative, zero, and positive regressions of the activity traits on body weight, the net effect of which was to produce overall independence of body weight from physical activity. We also demonstrated that the relQTLs produced these varying associations through differential epistatic interactions with a number of other epistatic QTLs throughout the genome.

Conclusion

It was concluded that individuals with specific combinations of genotypes at the relQTLs and epiQTLs might account for some of the variation typically seen in plots of the association of physical activity with body weight.     

Genetic dissection of testicular weight in the mouse with the BXD recombinant inbred strains

Testicular weights were studied in the mouse BXD recombinant inbred (RI) strains. These strains were derived from DBA/2J and C57BL/6J progenitors that differ significantly in their testicular weights (0.224 g ± 0.015 vs. 0.161 g ± 0.03, P < 0.0001). The heritability of testicular weights was calculated to be 0.53, and the minimum number of responsible effective factors was estimated to be 5.7. The total genome scanning of the BXD RI strains with over 1000 markers revealed a quantitative trait locus (QTL) on mouse Chromosome (Chr) 13 near the D13Mit3 marker (LOD score 6.9). This QTL region was designated Twq1 and associated with over 75% of genetic variability. Received: 23 January 1998 / Accepted: 16 March 1998