Quantitative trait locus mapping for seed mineral concentrations in two Arabidopsis thaliana recombinant inbred populations

Biofortification of foods, achieved by increasing the concentrations of minerals such as iron (Fe) and zinc (Zn), is a goal of plant scientists. Understanding genes that influence seed mineral concentration in a model plant such as Arabidopsis could help in the development of nutritionally enhanced crop cultivars. Quantitative trait locus (QTL) mapping for seed concentrations of calcium (Ca), copper (Cu), Fe, potassium (K), magnesium (Mg), manganese (Mn), phosphorus (P), sulfur (S), and Zn was performed using two recombinant inbred line (RIL) populations, Columbia (Col) x Landsberg erecta (Ler) and Cape Verde Islands (Cvi) x Ler, grown on multiple occasions. QTL mapping was also performed using data from silique hulls and the ratio of seed:hull mineral concentration of the Cvi x Ler population. Over 100 QTLs that affected seed mineral concentration were identified. Twenty-nine seed QTLs were found in more than one experiment, and several QTLs were found for both seed and hull mineral traits. A number of candidate genes affecting seed mineral concentration are discussed. These results indicate that A. thaliana is a suitable and convenient model for discovery of genes that affect seed mineral concentration. Some strong QTLs had no obvious candidate genes, offering the possibility of identifying unknown genes that affect mineral uptake and translocation to seeds.     

Genetics of quantitative traits in Arabidopsis thaliana

The genetic control of 22 quantitative traits, including developmental rates and sizes, was examined in generations of Arabidopsis thaliana derived from the cross between the ecotypes, Columbia (Col) and Landsberg erecta (Ler). The data were obtained from three sets of families raised in the same trial: the 16 basic generations, that is, parents, F(1)'s, F(2)'s, backcrosses, recombinant inbred lines (RILs) and a triple test cross (TTC), the latter produced by crossing the RILs to Col, Ler and their F(1). The data were analysed by two approaches. The first (approach A) involved traditional generation mean and variance component analysis and the second (B), based around the RILs and TTC families, involved marker-based QTL analysis.From (A), genetic differences between Col and Ler were detected for all traits with moderate heritabilities. Height at flowering was the only trait to show heterosis. Dominance was partial to complete for all height traits, and there was no overdominance but there was strong evidence for directional dominance. For most other traits, dominance was ambidirectional and incomplete, with average dominance ratios of around 80%. Epistasis, particularly of the duplicate type that opposes dominance, was a common feature of all traits. The presence of epistasis must imply multiple QTL for all traits.The QTL analysis located 38 significant effects in four regions of chromosomes I, II, IV and V, but not III. QTL affecting rosette size and leaf number were identified in all four regions, with days to maturity on chromosomes IV and V. The only QTL for height was located at the expected position of the erecta gene (chromosome II; 50 cM), but the additive and dominance effects of this single QTL did not adequately explain the generation means. The possible involvement of other interacting height QTL is discussed.     

Quantitative trait locus analysis of growth-related traits in a new Arabidopsis recombinant inbred population

Arabidopsis natural variation was used to analyze the genetics of plant growth rate. Screening of 22 accessions revealed a large variation for seed weight, plant dry weight and relative growth rate but not for water content. A positive correlation was observed between seed weight and plant area 10 d after planting, suggesting that seed weight affects plant growth during early phases of development. During later stages of plant growth this correlation was not significant, indicating that other factors determine growth rate during this phase. Quantitative trait locus (QTL) analysis, using 114 (F9 generation) recombinant inbred lines derived from the cross between Landsberg erecta (Ler, from Poland) and Shakdara (Sha, from Tadjikistan), revealed QTLs for seed weight, plant area, dry weight, relative growth rate, chlorophyll fluorescence, flowering time, and flowering-related traits. Growth traits (plant area, dry weight, and relative growth rate) colocated at five genomic regions. At the bottom of chromosome 5, colocation was found of QTLs for leaf area, leaf initiation speed, specific leaf area, and chlorophyll fluorescence but not for dry weight, indicating that this locus might be involved in leaf development. No consistent relation between growth traits and flowering time was observed despite some colocations. Some of the QTLs detected for flowering time overlapped with loci detected in other recombinant inbred line populations, but also new loci were identified. This study shows that Arabidopsis can successfully be used to study the genetic basis of complex traits like plant growth rate.     

Quantitative trait loci mapping in five new large recombinant inbred line populations of Arabidopsis thaliana genotyped with consensus single-nucleotide polymorphism markers

Quantitative approaches conducted in a single mapping population are limited by the extent of genetic variation distinguishing the parental genotypes. To overcome this limitation and allow a more complete dissection of the genetic architecture of complex traits, we built an integrated set of 15 new large Arabidopsis thaliana recombinant inbred line (RIL) populations optimized for quantitative trait loci (QTL) mapping, having Columbia as a common parent crossed to distant accessions. Here we present 5 of these populations that were validated by investigating three traits: flowering time, rosette size, and seed production as an estimate of fitness. The large number of RILs in each population (between 319 and 377 lines) and the high density of evenly spaced genetic markers scored ensure high power and precision in QTL mapping even under a minimal phenotyping framework. Moreover, the use of common markers across the different maps allows a direct comparison of the QTL detected within the different RIL sets. In addition, we show that following a selective phenotyping strategy by performing QTL analyses on genotypically chosen subsets of 164 RILs (core populations) does not impair the power of detection of QTL with phenotypic contributions >7%.     

Estimation of variance components of quantitative traits in inbred populations

Use of variance-component estimation for mapping of quantitative-trait loci in humans is a subject of great current interest. When only trait values, not genotypic information, are considered, variance-component estimation can also be used to estimate heritability of a quantitative trait. Inbred pedigrees present special challenges for variance-component estimation. First, there are more variance components to be estimated in the inbred case, even for a relatively simple model including additive, dominance, and environmental effects. Second, more identity coefficients need to be calculated from an inbred pedigree in order to perform the estimation, and these are computationally more difficult to obtain in the inbred than in the outbred case. As a result, inbreeding effects have generally been ignored in practice. We describe here the calculation of identity coefficients and estimation of variance components of quantitative traits in large inbred pedigrees, using the example of HDL in the Hutterites. We use a multivariate normal model for the genetic effects, extending the central-limit theorem of Lange to allow for both inbreeding and dominance under the assumptions of our variance-component model. We use simulated examples to give an indication of under what conditions one has the power to detect the additional variance components and to examine their impact on variance-component estimation. We discuss the implications for mapping and heritability estimation by use of variance components in inbred populations.     

QTL analysis of Al tolerance in recombinant inbred lines of Arabidopsis thaliana

Quantitative trait loci (QTLs) and epistasis for Arabidopsis thaliana aluminum (Al) tolerance were analyzed using a recombinant inbred (RI) population of 100 lines derived from a cross between Landsberg erecta and Columbia (Col). Root growth of the RI population was determined in hydroponics using solutions containing 0 or 4 micro M of AlCl(3 )and a series of nutrients, except P(i), at pH 5.0. Al tolerance was defined as relative root length [RRL: plus Al/minus Al (%)], and the RI lines ranged from 22.6 to 97.4% with a broad sense heritability of 0.99. Using the composite interval mapping method, two significant single factor QTLs (P<0.05) were detected by RRL on chromosomes 1 and 4, where the Col allele showed positive and negative effects on the Al tolerance. These QTLs could explain about 43% of the total variation of Al tolerance among the RI population. On the other hand, five epistatic loci pairs were identified by the complete pair-wise search method (P<0.0005). No single factor QTL and epistatic loci pairs were shared by the root length in the control and the RRL, suggesting that the loci identified by the RRL would be specific for Al treatment and controlling Al tolerance among the RI population.     

Identification of quantitative trait loci across recombinant inbred lines and testcross populations for traits of agronomic importance in rice

This study was conducted to determine whether quantitative trait loci (QTL) controlling traits of agronomic importance detected in recombinant inbred lines (RILs) are also expressed in testcross (TC) hybrids of rice. A genetic map was constructed using an RIL population derived from a cross between B5 and Minghui 63, a parent of the most widely grown hybrid rice cultivar in China. Four TC hybrid populations were produced by crossing the RILs with three maintaining lines for the widely used cytoplasmic male-sterile (CMS) lines and the genic male-sterile line Peiai64s. The mean values of the RILs for the seven traits investigated were significantly correlated to those of the F1 hybrids in the four TC populations. Twenty-seven main-effect QTL were identified in the RILs. Of these, the QTL that had the strongest effect on each of the seven traits in the RILs was detected in two or more of the TC populations, and six other QTL were detected in one TC population. Epistatic analysis revealed that the effect of epistatic QTL was relatively weak and cross combination specific. Searching publicly available QTL data in rice revealed the positional convergence of the QTL with the strongest effect in a wide range of populations and under different environments. Since the main-effect QTL is expressed across different testers, and in different genetic backgrounds and environments, it is a valuable target for gene manipulation and for further application in rice breeding. When a restorer line that expresses main-effect QTL is bred, it could be used in a number of cross combinations.     

Heterosis for biomass-related traits in Arabidopsis investigated by quantitative trait loci analysis of the triple testcross design with recombinant inbred lines

Arabidopsis thaliana has emerged as a leading model species in plant genetics and functional genomics including research on the genetic causes of heterosis. We applied a triple testcross (TTC) design and a novel biometrical approach to identify and characterize quantitative trait loci (QTL) for heterosis of five biomass-related traits by (i) estimating the number, genomic positions, and genetic effects of heterotic QTL, (ii) characterizing their mode of gene action, and (iii) testing for presence of epistatic effects by a genomewide scan and marker x marker interactions. In total, 234 recombinant inbred lines (RILs) of Arabidopsis hybrid C24 x Col-0 were crossed to both parental lines and their F1 and analyzed with 110 single-nucleotide polymorphism (SNP) markers. QTL analyses were conducted using linear transformations Z1, Z2, and Z3 calculated from the adjusted entry means of TTC progenies. With Z1, we detected 12 QTL displaying augmented additive effects. With Z2, we mapped six QTL for augmented dominance effects. A one-dimensional genome scan with Z3 revealed two genomic regions with significantly negative dominance x additive epistatic effects. Two-way analyses of variance between marker pairs revealed nine digenic epistatic interactions: six reflecting dominance x dominance effects with variable sign and three reflecting additive x additive effects with positive sign. We conclude that heterosis for biomass-related traits in Arabidopsis has a polygenic basis with overdominance and/or epistasis being presumably the main types of gene action.     

New Arabidopsis recombinant inbred line populations genotyped using SNPWave and their use for mapping flowering-time quantitative trait loci

The SNPWave marker system, based on SNPs between the reference accessions Colombia-0 and Landsberg erecta (Ler), was used to distinguish a set of 92 Arabidopsis accessions from various parts of the world. In addition, we used these markers to genotype three new recombinant inbred line populations for Arabidopsis, having Ler as a common parent that was crossed with the accessions Antwerp-1, Kashmir-2, and Kondara. The benefit of using multiple populations that contain many similar markers and the fact that all markers are linked to the physical map of Arabidopsis facilitates the quantitative comparison of maps. Flowering-time variation was analyzed in the three recombinant inbred line populations. Per population, four to eight quantitative trait loci (QTL) were detected. The comparison of the QTL positions related to the physical map allowed the estimate of 12 different QTL segregating for flowering time for which Ler has an allele different from one, two, or three of the other accessions.     

Isolation and mapping of a new set of 129 RFLP markers in Arabidopsis thaliana using recombinant inbred lines

A new collection of 129 Arabidopsis thaliana RFLP markers has been established based upon DNA fragments cloned in the pUC119 plasmid vector and insert end sequences of P1 clones. Dominant/null alleles affecting low-copy number sequences account for nine of the mapped polymorphisms, suggesting that deletions are not rare in A. thaliana . Recombinant inbred (RI) lines were used for mapping these marker loci. RI line-based mapping allows integration of this set of markers with markers previously reported as well as with any markers mapped in the future using this replenishable mapping resource. These markers are useful for map-based gene isolation and genome physical mapping in A. thaliana as well as studies of chromosome colinearity (synteny) with related species.     

Epistasis for fitness-related quantitative traits in Arabidopsis thaliana grown in the field and in the greenhouse

The extent to which epistasis contributes to adaptation, population differentiation, and speciation is a long-standing and important problem in evolutionary genetics. Using recombinant inbred (RI) lines of Arabidopsis thaliana grown under natural field conditions, we have examined the genetic architecture of fitness-correlated traits with respect to epistasis; we identified both single-locus additive and two-locus epistatic QTL for natural variation in fruit number, germination, and seed length and width. For fruit number, we found seven significant epistatic interactions, but only two additive QTL. For seed germination, length, and width, there were from two to four additive QTL and from five to eight epistatic interactions. The epistatic interactions were both positive and negative. In each case, the magnitude of the epistatic effects was roughly double that of the effects of the additive QTL, varying from -41% to +29% for fruit number and from -5% to +4% for seed germination, length, and width. A number of the QTL that we describe participate in more than one epistatic interaction, and some loci identified as additive also may participate in an epistatic interaction; the genetic architecture for fitness traits may be a network of additive and epistatic effects. We compared the map positions of the additive and epistatic QTL for germination, seed width, and seed length from plants grown in both the field and the greenhouse. While the total number of significant additive and epistatic QTL was similar under the two growth conditions, the map locations were largely different. We found a small number of significant epistatic QTL x environment effects when we tested directly for them. Our results support the idea that epistatic interactions are an important part of natural genetic variation and reinforce the need for caution in comparing results from greenhouse-grown and field-grown plants.     

Mapping quantitative trait loci using the experimental designs of recombinant inbred populations

In the data collection of the QTL experiments using recombinant inbred (RI) populations, when individuals are genotyped for markers in a population, the trait values (phenotypes) can be obtained from the genotyped individuals (from the same population) or from some progeny of the genotyped individuals (from the different populations). Let Fu be the genotyped population and Fv (v>or=u) be the phenotyped population. The experimental designs that both marker genotypes and phenotypes are recorded on the same populations can be denoted as (Fu/Fv, u=v) designs and that genotypes and phenotypes are obtained from the different populations can be denoted as (Fu/Fv, v>u) designs. Although most of the QTL mapping experiments have been conducted on the backcross and F2(F2/F2) designs, the other (Fu/Fv, v>or=u) designs are also very popular. The great benefits of using the other (Fu/Fv, v>or=u) designs in QTL mapping include reducing cost and environmental variance by phenotyping several progeny for the genotyped individuals and taking advantages of the changes in population structures of other RI populations. Current QTL mapping methods including those for the (Fu/Fv, u=v) designs, mostly for the backcross or F2/F2 design, and for the F2/F3 design based on a one-QTL model are inadequate for the investigation of the mapping properties in the (Fu/Fv, uor=u) designs. In addition, the QTL mapping properties of the proposed and approximate methods in different designs are discussed. Simulations were performed to evaluate the performance of the proposed and approximate methods. The proposed method is proven to be able to correct the problems of the approximate and current methods for improving the resolution of genetic architecture of quantitative traits and can serve as an effective tool to explore the QTL mapping study in the system of RI populations.     

Variance for water-use efficiency among ecotypes and recombinant inbred lines of Arabidopsis thaliana (Brassicaceae)

Knowledge of natural variation among ecotypes and recombinant inbred lines of Arabidopsis thaliana L. Heynh for season-long water-use efficiency (WUE, moles of carbon accumulated per mole of water used) is useful in the design of experiments to understand the genetic control of this phenomenon. Water-use efficiency among 31 container-grown Arabidopsis ecotypes ranged from a high of 2.40 to a low of 1.86 mg/g (grams of dry aerial biomass per gram of water used). Genetic variance for WUE was observed among 80 F₅ recombinant inbred lines (RILs) derived from a cross between the highest (Lip-0) by the lowest (Edi-0) ecotype. The heritability of WUE was calculated as 0.18 ± 0.07. The mean WUE for the ten highest and ten lowest RILs was 2.42 and 1.05 mg/g, respectively. A significant difference was observed in the composition of stable isotopes of plant carbon (δ¹³C) between the mean of the ten highest (-31.23±) and the mean of the ten lowest (-31.96±) RILs based on WUE. Characterization of the 80 RILs provides a structured population for further genetic and physiologic study of WUE.     

Integration of CAPS markers into the RFLP map generated using recombinant inbred lines of Arabidopsis thaliana

Konieczny and Ausubel have described a technique whereby Arabidopsis thaliana loci can be rapidly mapped to one of the ten chromosome arms using a small number of F2 progeny from crosses between the ecotypes Landsberg erecta and Columbia. The technique involves the use of 18 co-dominant, cleaved amplified polymorphic sequence (CAPS) markers which are evenly distributed throughout the Arabidopsis genome. We have mapped these 18 markers using recombinant inbred (RI) lines generated in our laboratory. These data enable a better integration of loci mapped relative to the CAPS markers into the restriction fragment length polymorphism (RFLP) map generated using Arabidopsis RI lines.     

Use of recombinant inbred lines of wheat for study of associations of high-molecular-weight glutenin subunit alleles to quantitative traits

Summary Recombinant inbred lines (RILs) derived by single plant descent to F₈ from a hybrid of Anza, a low-quality cultivar, and Cajeme 71, a high-quality cultivar, differed in alleles at three high-molecular-weight glutenin (HMW-glu) seed storage protein loci. The 48 RILs were classified by SDS-PAGE for the Anza alleles Glu-Alc (null), Glu-B1b (subunits 7 + 8), and Glu-D1a (subunits 2 + 12) and for Cajeme 71 alleles Glu-A1a (sub-unit 1), Glu-B1I (subunits 17 + 18), and Glu-D1d (subunits 5 + 10). All RILs and parents were grown in a replicated field trial with three levels of nitrogen (N) fertilization. Additive and additive x additive gene effects for the three loci were detected by orthogonal comparisons of means for each of six wheat end-use quality traits. Each HMW-glu genotype was represented by three to ten RILs so that variability among RILs within each HMW-glu genotype could be examined. N effects were consistently small. All traits except flour yield were highly correlated with predictor traits studied earlier. Flour protein content, baking water absorption, dough mixing time, bread loaf volume, and bread loaf crumb score were all correlated, suggesting similar gene control for these traits; however, specific additive locus contributions were evident: _B for flour yield; _B and _D for flour protein; and _B for absorption, but differing in sign; all three loci for mixing time, but _B was negative; and all three loci were positively associated with loaf volume. Digenic epistatic effects were significant for flour yield (_AD), flour protein (_AB), and absorption and mixing time (_AD, _BD). Only flour yield showed a trigenic epistatic effect. Six of seven epistatic effects were negative, thus showing how progress in breeding for high quality may be impeded by interaction of genes which, by themselves, have strong positive additive effects. Considerable genetic variance among RILs within a HMW-glu genotype was detected for all traits, and the summation of effects accounted for a mean of 13% of the parental differences for the six traits examined in this study. Clearly, further resolution of the genetics of wheat quality would be desirable from a plant breeding point of view.     

Use of recombinant inbred lines of wheat for study of associations of high-molecular-weight glutenin subunit alleles to quantitative traits

Summary The high-molecular-weight glutenin subunits (HMW glutenin), encoded by alleles at homoeologous lociGlu-A1,Glu-B1, andGlu-D1 on the long arms of chromosomes1A,1B, and1D of a set of F₈ random recombinant inbred lines (RIL) derived from the bread wheat cross Anza × Cajeme 71, were classified by SDS-PAGE. Anza has poor breadmaking quality and HMW-glutenin subunits (Payne numbers) null (Glu-A1c), 7+8 (Glu-B1b), and 2+12 (Glu-D1a); Cajeme 71 has good quality and 1 (Glu-A1a), 17+18 (Glu-B1i), and 5+10 (Glu-D1d). The combinations of these alleles in the RIL were examined for associations with grain yield and four indicators of grain quality — protein content, yellowberry, pearling index, and SDS sedimentation volume. Data were obtained from a field experiment with three nitrogen fertilization treatments on 48 RIL and the parents. Orthogonal partitioning of the genetic variance associated with the three HMW glutenin subunit loci into additive and epistatic (digenic and trigenic) effects showed strong associations of these loci with grain yield and the indicators of quality; however, the associations accounted for no more than 25% of the differences between the parents. Genetic variance was detected among the RIL, which had the same HMW glutenin genotype for all traits. Epistatic effects were absent for grain yield and yellowberry, but were substantial for grain protein content, pearling index, and SDS sedimentation volume. All three loci had large single-locus additive effects for grain yield, protein, and SDS sedimentation volume. Yellowberry was largely influenced byGlu-B1 andGlu-D1, whereas pearling index was associated withGlu-A1 andGlu-B1. Even though the observed associations-of effects of HMW glutenin loci with the quantitative characters were small relative to the total genetic variability, they are of considerable importance in understanding the genetics of wheat quality, and are useful in the development of new wheat varieties with specific desired characteristics.     

Quantitative trait loci controlling resistance to cadmium rhizotoxicity in two recombinant inbred populations of Arabidopsis thaliana are partially shared by those for hydrogen peroxide resistance

To understand mechanisms of cadmium (Cd) tolerance variation associated with root elongation in Arabidopsis thaliana , quantitative trait loci (QTLs) and epistasis were analyzed using relative root length (RRL: % of the root length in +Cd to −Cd) as a tolerant index. Using the composite interval mapping method, three major QTLs ( P < 0.05) were detected on chromosomes 2, 4 and 5 in the recombinant inbred population derived from a cross between Landsberg erecta (L er −0) and Columbia (Col-4). The highest logarithm of odds (LOD) of 5.6 was detected with the QTL on chromosome 5 (QTL5), which is linked to the genetic marker CDPK9 and explained about 26% of the Cd tolerance variation. There was no significant difference in Cd-translocation ratio from roots to shoots between tolerant and sensitive recombinant inbreed lines (RILs), while greater accumulations of reactive oxygen species were observed in the roots of sensitive RILs. This suggested that accumulation of ROS would explain Cd tolerance variation of the L er /Col RILs, which is mainly controlled by the QTL on chromosome 5. The QTL5 in L er /Col population was also detected as one of the major QTLs controlling tolerances to hydrogen peroxide and to copper, which is another ROS generating rhizotoxic metal. The same chromosomal position was detected as a common major QTL for Cd and hydrogen peroxide tolerances in a different recombinant inbreed (RI) population derived from a cross of Col- gl1 and Kashmir (Kas-1). These data, along with a multitraits QTL analysis in both sets of RILs, suggest that peroxide damage depends on the genotype at a major Cd-tolerant locus on the upper part of chromosome 5.     

Comparison between marker-assisted selection and phenotypical selection in a set of Arabidopsis thaliana recombinant inbred lines

 Parents were selected from a well-characterised Arabidopsis recombinant inbred line (RIL) population based on (1) their phenotype for flowering time or (2) marker and QTL information that had been assessed previously. The F₂ offspring obtained from pairs of selected RILs was analysed for these traits, and the results obtained with these two methods of selection were compared. Selection based on marker and QTL information gave approximately the same result as selection based on phenotype. The relative high heritability of flowering time in Arabidopsis facilitated successful phenotypical selection. The difference in selection result that was anticipated to be in favour of the marker-assisted approach was therefore not observed. Received: 29 November 1997 / Accepted: 8 June 1998     

Seasonal and plant-density dependency for quantitative trait loci affecting flowering time in multiple populations of Arabidopsis thaliana

Multiple environmental cues regulate the transition to flowering. In natural environments, plants perceive seasonal progression by changes in day length and growth temperature, and plant density is monitored by changes in the light quality reflected from neighbouring vegetation. To understand the seasonal and plant-density dependence associated with natural allelic variation in flowering time, we conducted a quantitative trait loci (QTL) mapping study in Ler x Cvi, Bay x Sha and Ler x No-0 recombinant inbred line (RIL) populations of Arabidopsis thaliana. Days and total leaf number to bolting were examined under low and high plant density (200 or 1600 plants m(-2)) in autumn-winter and spring seasons. We found between 4 and 10 QTLs associated with seasonal and density variations in each RIL population. For Ler x Cvi and Bay x Sha RIL populations, a major proportion of QTLs showed seasonal and density interaction (up to 63%) and four QTLs were common to all environments (21%). Only three QTLs showed seasonal or density dependency. By aligning the linkage maps onto a common physical map, we detected at least one QTL at chromosome 2 and two QTLs at chromosome 5 that overlap between the three RIL populations, suggesting that these QTLs play a crucial role in the adaptive control of flowering time.