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1.  Variation in Seed Dormancy Quantitative Trait Loci in Arabidopsis thaliana Originating from One Site 
PLoS ONE  2011;6(6):e20886.
A Quantitative Trait Locus (QTL) analysis was performed using two novel Recombinant Inbred Line (RIL) populations, derived from the progeny between two Arabidopsis thaliana genotypes collected at the same site in Kyoto (Japan) crossed with the reference laboratory strain Landsberg erecta (Ler). We used these two RIL populations to determine the genetic basis of seed dormancy and flowering time, which are assumed to be the main traits controlling life history variation in Arabidopsis. The analysis revealed quantitative variation for seed dormancy that is associated with allelic variation at the seed dormancy QTL DOG1 (for Delay Of Germination 1) in one population and at DOG6 in both. These DOG QTL have been previously identified using mapping populations derived from accessions collected at different sites around the world. Genetic variation within a population may enhance its ability to respond accurately to variation within and between seasons. In contrast, variation for flowering time, which also segregated within each mapping population, is mainly governed by the same QTL.
doi:10.1371/journal.pone.0020886
PMCID: PMC3127951  PMID: 21738591
2.  Natural Genetic Variation in Arabidopsis: Tools, Traits and Prospects for Evolutionary Ecology 
Annals of Botany  2007;99(6):1043-1054.
Background
The model plant Arabidopsis thaliana (Arabidopsis) shows a wide range of genetic and trait variation among wild accessions. Because of its unparalleled biological and genomic resources, the potential of Arabidopsis for molecular genetic analysis of this natural variation has increased dramatically in recent years.
Scope
Advanced genomics has accelerated molecular phylogenetic analysis and gene identification by quantitative trait loci (QTL) mapping and/or association mapping in Arabidopsis. In particular, QTL mapping utilizing natural accessions is now becoming a major strategy of gene isolation, offering an alternative to artificial mutant lines. Furthermore, the genomic information is used by researchers to uncover the signature of natural selection acting on the genes that contribute to phenotypic variation. The evolutionary significance of such genes has been evaluated in traits such as disease resistance and flowering time. However, although molecular hallmarks of selection have been found for the genes in question, a corresponding ecological scenario of adaptive evolution has been difficult to prove. Ecological strategies, including reciprocal transplant experiments and competition experiments, and utilizing near-isogenic lines of alleles of interest will be a powerful tool to measure the relative fitness of phenotypic and/or allelic variants.
Conclusions
As the plant model organism, Arabidopsis provides a wealth of molecular background information for evolutionary genetics. Because genetic diversity between and within Arabidopsis populations is much higher than anticipated, combining this background information with ecological approaches might well establish Arabidopsis as a model organism for plant evolutionary ecology.
doi:10.1093/aob/mcl281
PMCID: PMC3243570  PMID: 17259228
Arabidopsis; natural genetic variation; natural trait variation; QTL mapping; LD mapping; plant development; plant evolution; molecular ecology
3.  Metabolite profiling and quantitative genetics of natural variation for flavonoids in Arabidopsis  
Journal of Experimental Botany  2012;63(10):3749-3764.
Little is known about the range and the genetic bases of naturally occurring variation for flavonoids. Using Arabidopsis thaliana seed as a model, the flavonoid content of 41 accessions and two recombinant inbred line (RIL) sets derived from divergent accessions (Cvi-0×Col-0 and Bay-0×Shahdara) were analysed. These accessions and RILs showed mainly quantitative rather than qualitative changes. To dissect the genetic architecture underlying these differences, a quantitative trait locus (QTL) analysis was performed on the two segregating populations. Twenty-two flavonoid QTLs were detected that accounted for 11–64% of the observed trait variations, only one QTL being common to both RIL sets. Sixteen of these QTLs were confirmed and coarsely mapped using heterogeneous inbred families (HIFs). Three genes, namely TRANSPARENT TESTA (TT)7, TT15, and MYB12, were proposed to underlie their variations since the corresponding mutants and QTLs displayed similar specific flavonoid changes. Interestingly, most loci did not co-localize with any gene known to be involved in flavonoid metabolism. This latter result shows that novel functions have yet to be characterized and paves the way for their isolation.
doi:10.1093/jxb/ers067
PMCID: PMC3388840  PMID: 22442426
Arabidopsis; flavonoids; metabolite profiling; natural variation; quantitative trait loci
4.  QTL Mapping in New Arabidopsis thaliana Advanced Intercross-Recombinant Inbred Lines 
PLoS ONE  2009;4(2):e4318.
Background
Even when phenotypic differences are large between natural or domesticated strains, the underlying genetic basis is often complex, and causal genomic regions need to be identified by quantitative trait locus (QTL) mapping. Unfortunately, QTL positions typically have large confidence intervals, which can, for example, lead to one QTL being masked by another, when two closely linked loci are detected as a single QTL. One strategy to increase the power of precisely localizing small effect QTL, is the use of an intercross approach before inbreeding to produce Advanced Intercross RILs (AI-RILs).
Methodology/Principal Findings
We present two new AI-RIL populations of Arabidopsis thaliana genotyped with an average intermarker distance of 600 kb. The advanced intercrossing design led to expansion of the genetic map in the two populations, which contain recombination events corresponding to 50 kb/cM in an F2 population. We used the AI-RILs to map QTL for light response and flowering time, and to identify segregation distortion in one of the AI-RIL populations due to a negative epistatic interaction between two genomic regions.
Conclusions/Significance
The two new AI-RIL populations, EstC and KendC, derived from crosses of Columbia (Col) to Estland (Est-1) and Kendallville (Kend-L) provide an excellent resource for high precision QTL mapping. Moreover, because they have been genotyped with over 100 common markers, they are also excellent material for comparative QTL mapping.
doi:10.1371/journal.pone.0004318
PMCID: PMC2629843  PMID: 19183806
5.  Identification of metabolic and biomass QTL in Arabidopsis thaliana in a parallel analysis of RIL and IL populations 
The Plant Journal   2008;53(6):960-972.
Plant growth and development are tightly linked to primary metabolism and are subject to natural variation. In order to obtain an insight into the genetic factors controlling biomass and primary metabolism and to determine their relationships, two Arabidopsis thaliana populations [429 recombinant inbred lines (RIL) and 97 introgression lines (IL), derived from accessions Col-0 and C24] were analyzed with respect to biomass and metabolic composition using a mass spectrometry-based metabolic profiling approach. Six and 157 quantitative trait loci (QTL) were identified for biomass and metabolic content, respectively. Two biomass QTL coincide with significantly more metabolic QTL (mQTL) than statistically expected, supporting the notion that the metabolic profile and biomass accumulation of a plant are linked. On the same basis, three out the six biomass QTL can be simulated purely on the basis of metabolic composition. QTL based on analysis of the introgression lines were in substantial agreement with the RIL-based results: five of six biomass QTL and 55% of the mQTL found in the RIL population were also found in the IL population at a significance level of P ≤ 0.05, with >80% agreement on the allele effects. Some of the differences could be attributed to epistatic interactions. Depending on the search conditions, metabolic pathway-derived candidate genes were found for 24–67% of all tested mQTL in the database AraCyc 3.5. This dataset thus provides a comprehensive basis for the detection of functionally relevant variation in known genes with metabolic function and for identification of genes with hitherto unknown roles in the control of metabolism.
doi:10.1111/j.1365-313X.2007.03383.x
PMCID: PMC2268983  PMID: 18047556
metabolic quantitative trait loci (mQTL); recombinant inbred line (RIL); introgression line (IL); Arabidopsis; GC-MS; metabolomics
6.  Natural Variation in Partial Resistance to Pseudomonas syringae Is Controlled by Two Major QTLs in Arabidopsis thaliana 
PLoS ONE  2006;1(1):e123.
Background
Low-level, partial resistance is pre-eminent in natural populations, however, the mechanisms underlying this form of resistance are still poorly understood.
Methodology/Principal Findings
In the present study, we used the model pathosystem Pseudomonas syringae pv. tomato DC3000 (Pst) - Arabidopsis thaliana to study the genetic basis of this form of resistance. Phenotypic analysis of a set of Arabidopsis accessions, based on evaluation of in planta pathogen growth revealed extensive quantitative variation for partial resistance to Pst. It allowed choosing a recombinant inbred line (RIL) population derived from a cross between the accessions Bayreuth and Shahdara for quantitative genetic analysis. Experiments performed under two different environmental conditions led to the detection of two major and two minor quantitative trait loci (QTLs) governing partial resistance to Pst and called PRP-Ps1 to PRP-Ps4. The two major QTLs, PRP-Ps1 and PRP-Ps2, were confirmed in near isogenic lines (NILs), following the heterogeneous inbred families (HIFs) strategy. Analysis of marker gene expression using these HIFs indicated a negative correlation between the induced amount of transcripts of SA-dependent genes PR1, ICS and PR5, and the in planta bacterial growth in the HIF segregating at PRP-Ps2 locus, suggesting an implication of PRP-Ps2 in the activation of SA dependent responses.
Conclusions/Significance
These results show that variation in partial resistance to Pst in Arabidopsis is governed by relatively few loci, and the validation of two major loci opens the way for their fine mapping and their cloning, which will improve our understanding of the molecular mechanisms underlying partial resistance.
doi:10.1371/journal.pone.0000123
PMCID: PMC1762404  PMID: 17205127
7.  Natural Variation in Seed Very Long Chain Fatty Acid Content Is Controlled by a New Isoform of KCS18 in Arabidopsis thaliana 
PLoS ONE  2012;7(11):e49261.
Oil from oleaginous seeds is mainly composed of triacylglycerols. Very long chain fatty acids (VLCFAs) are major constituents of triacylglycerols in many seed oils and represent valuable feedstock for industrial purposes. To identify genetic factors governing natural variability in VLCFA biosynthesis, a quantitative trait loci (QTL) analysis using a recombinant inbred line population derived from a cross between accessions Bay-0 and Shahdara was performed in Arabidopsis thaliana. Two fatty acid chain length ratio (CLR) QTL were identified, with one major locus, CLR.2, accounting for 77% of the observed phenotypic variation. A fine mapping and candidate gene approach showed that a key enzyme of the fatty acid elongation pathway, the β-ketoacyl-CoA synthase 18 (KCS18), was responsible for the CLR.2 QTL detected between Bay-0 and Shahdara. Association genetics and heterologous expression in yeast cells identified a single point mutation associated with an alteration of KCS18 activity, uncovering the molecular bases for the modulation of VLCFA content in these two natural populations of Arabidopsis. Identification of this kcs18 mutant with altered activity opens new perspectives for the modulation of oil composition in crop plants.
doi:10.1371/journal.pone.0049261
PMCID: PMC3493540  PMID: 23145136
8.  Quantitative trait locus analysis of hybrid pedigrees: variance-components model, inbreeding parameter, and power 
BMC Genetics  2007;8:50.
Background
For the last years reliable mapping of quantitative trait loci (QTLs) has become feasible through linkage analysis based on the variance-components method. There are now many approaches to the QTL analysis of various types of crosses within one population (breed) as well as crosses between divergent populations (breeds). However, to analyse a complex pedigree with dominance and inbreeding, when the pedigree's founders have an inter-population (hybrid) origin, it is necessary to develop a high-powered method taking into account these features of the pedigree.
Results
We offer a universal approach to QTL analysis of complex pedigrees descended from crosses between outbred parental lines with different QTL allele frequencies. This approach improves the established variance-components method due to the consideration of the genetic effect conditioned by inter-population origin and inbreeding of individuals. To estimate model parameters, namely additive and dominant effects, and the allelic frequencies of the QTL analysed, and also to define the QTL positions on a chromosome with respect to genotyped markers, we used the maximum-likelihood method. To detect linkage between the QTL and the markers we propose statistics with a non-central χ2-distribution that provides the possibility to deduce analytical expressions for the power of the method and therefore, to estimate the pedigree's size required for 80% power. The method works for arbitrarily structured pedigrees with dominance and inbreeding.
Conclusion
Our method uses the phenotypic values and the marker information for each individual of the pedigree under observation as initial data and can be valuable for fine mapping purposes. The power of the method is increased if the QTL effects conditioned by inter-population origin and inbreeding are enhanced. Several improvements can be developed to take into account fixed factors affecting trait formation, such as age and sex.
doi:10.1186/1471-2156-8-50
PMCID: PMC2014774  PMID: 17651509
9.  Natural Variation in Arabidopsis thaliana Revealed a Genetic Network Controlling Germination Under Salt Stress 
PLoS ONE  2010;5(12):e15198.
Plant responses to environmental stresses are polygenic and complex traits. In this study quantitative genetics using natural variation in Arabidopsis thaliana was used to investigate the genetic architecture of plant responses to salt stress. Eighty seven A. thaliana accessions were screened and showed a large variation for root development and seed germination under 125 and 200 mM NaCl, respectively. Twenty two quantitative trait loci for these traits have been detected by phenotyping two recombinants inbred line populations, Sha x Col and Sha x Ler. Four QTLs controlling germination under salt were detected in the Sha x Col population. Interestingly, only one allelic combination at these four QTLs inhibits germination under salt stress, implying strong epistatic interactions between them. In this interacting context, we confirmed the effect of one QTL by phenotyping selected heterozygous inbred families. We also showed that this QTL is involved in the control of germination under other stress conditions such as KCl, mannitol, cold, glucose and ABA. Our data highlights the presence of a genetic network which consists of four interacting QTLs and controls germination under limiting environmental conditions.
doi:10.1371/journal.pone.0015198
PMCID: PMC3004798  PMID: 21188145
10.  Six new recombinant inbred populations for the study of quantitative traits in Arabidopsis thaliana 
Quantitative approaches are now widely used to study the genetic architecture of complex traits. However, most studies have been conducted in single mapping populations, which sample only a fraction of the natural allelic variation available within a gene pool and can identify only a subset of the loci controlling the traits. To enable the progress towards an understanding of the global genetic architecture of a broad range of complex traits, we have developed and characterised six new Arabidopsis thaliana recombinant inbred populations. To evaluate the utility of these populations for integrating analyses from multiple populations, we identified quantitative trait loci (QTL) controlling flowering time in vernalized plants growing in 16 h days. We used the physical positions of markers to align the linkage maps of our populations with those of six existing populations. We identified seven QTL in genomic locations coinciding with those identified in previous studies and in addition a further eight QTL were identified.
Electronic supplementary material
The online version of this article (doi:10.1007/s00122-007-0696-9) contains supplementary material, which is available to authorized users.
doi:10.1007/s00122-007-0696-9
PMCID: PMC2755751  PMID: 18193187
11.  The Flowering Repressor SVP Underlies a Novel Arabidopsis thaliana QTL Interacting with the Genetic Background 
PLoS Genetics  2013;9(1):e1003289.
The timing of flowering initiation is a fundamental trait for the adaptation of annual plants to different environments. Large amounts of intraspecific quantitative variation have been described for it among natural accessions of many species, but the molecular and evolutionary mechanisms underlying this genetic variation are mainly being determined in the model plant Arabidopsis thaliana. To find novel A. thaliana flowering QTL, we developed introgression lines from the Japanese accession Fuk, which was selected based on the substantial transgression observed in an F2 population with the reference strain Ler. Analysis of an early flowering line carrying a single Fuk introgression identified Flowering Arabidopsis QTL1 (FAQ1). We fine-mapped FAQ1 in an 11 kb genomic region containing the MADS transcription factor gene SHORT VEGETATIVE PHASE (SVP). Complementation of the early flowering phenotype of FAQ1-Fuk with a SVP-Ler transgen demonstrated that FAQ1 is SVP. We further proved by directed mutagenesis and transgenesis that a single amino acid substitution in SVP causes the loss-of-function and early flowering of Fuk allele. Analysis of a worldwide collection of accessions detected FAQ1/SVP-Fuk allele only in Asia, with the highest frequency appearing in Japan, where we could also detect a potential ancestral genotype of FAQ1/SVP-Fuk. In addition, we evaluated allelic and epistatic interactions of SVP natural alleles by analysing more than one hundred transgenic lines carrying Ler or Fuk SVP alleles in five genetic backgrounds. Quantitative analyses of these lines showed that FAQ1/SVP effects vary from large to small depending on the genetic background. These results support that the flowering repressor SVP has been recently selected in A. thaliana as a target for early flowering, and evidence the relevance of genetic interactions for the intraspecific evolution of FAQ1/SVP and flowering time.
Author Summary
In many plant species, the timing of flowering initiation shows abundant quantitative variation among natural varieties, which reflects the importance of this trait for adaptation to different environments. Currently, a major goal in plant biology is to determine the molecular and evolutionary bases of this natural genetic variation. In this study we demonstrate that the central flowering regulator SHORT VEGETATIVE PHASE (SVP), encoding a MADS transcription factor, is involved in the flowering natural variation of the model organism Arabidopsis thaliana. In particular, we prove that a structural change caused by a single amino acid substitution generates a SVP early flowering allele that is distributed only in Asia. Furthermore, genetic interactions have been shown to be a component of the natural variation for many important adaptive traits. However, very few studies, either in animals or plants, have systematically addressed the extent of genetic interactions among specific alleles responsible for the natural variation of complex traits. Our study shows that the flowering effects of SVP natural alleles depend significantly on the genetic background; and, subsequently, we demonstrate the relevance of epistasis for the evolution of this crucial transcription factor and flowering time.
doi:10.1371/journal.pgen.1003289
PMCID: PMC3561112  PMID: 23382706
12.  Genetic dissection of rice grain shape using a recombinant inbred line population derived from two contrasting parents and fine mapping a pleiotropic quantitative trait locus qGL7 
BMC Genetics  2010;11:16.
Background
The three-dimensional shape of grain, measured as grain length, width, and thickness (GL, GW, and GT), is one of the most important components of grain appearance in rice. Determining the genetic basis of variations in grain shape could facilitate efficient improvements in grain appearance. In this study, an F7:8 recombinant inbred line population (RIL) derived from a cross between indica and japonica cultivars (Nanyangzhan and Chuan7) contrasting in grain size was used for quantitative trait locus (QTL) mapping. A genetic linkage map was constructed with 164 simple sequence repeat (SSR) markers. The major aim of this study was to detect a QTL for grain shape and to fine map a minor QTL, qGL7.
Results
Four QTLs for GL were detected on chromosomes 3 and 7, and 10 QTLs for GW and 9 QTLs for GT were identified on chromosomes 2, 3, 5, 7, 9 and 10, respectively. A total of 28 QTLs were identified, of which several are reported for the first time; four major QTLs and six minor QTLs for grain shape were also commonly detected in both years. The minor QTL, qGL7, exhibited pleiotropic effects on GL, GW, GT, 1000-grain weight (TGW), and spikelets per panicle (SPP) and was further validated in a near isogenic F2 population (NIL-F2). Finally, qGL7 was narrowed down to an interval between InDel marker RID711 and SSR marker RM6389, covering a 258-kb region in the Nipponbare genome, and cosegregated with InDel markers RID710 and RID76.
Conclusion
Materials with very different phenotypes were used to develop mapping populations to detect QTLs because of their complex genetic background. Progeny tests proved that the minor QTL, qGL7, could display a single mendelian characteristic. Therefore, we suggested that minor QTLs for traits with high heritability could be isolated using a map-based cloning strategy in a large NIL-F2 population. In addition, combinations of different QTLs produced diverse grain shapes, which provide the ability to breed more varieties of rice to satisfy consumer preferences.
doi:10.1186/1471-2156-11-16
PMCID: PMC2846863  PMID: 20184774
13.  Molecular marker genotypes, heterozygosity and genetic interactions explain heterosis in Arabidopsis thaliana 
Heredity  2005;94(3):295-304.
The underlying mechanisms for hybrid vigor or heterosis are elusive. Here we report a population of recombinant inbred lines (RILs), derived from the two ecotypes, Col and Ler, which can serve as a permanent resource for studying the molecular basis of hybrid vigor in Arabidopsis. Using a North Carolina mating design III (NCIII), we determined the additive and dominant nature of gene action in this population. We detected heterosis among crosses of RILs with one of the two parents (Col and Ler) and analyzed genotypes and heterozygosities for RILs and test cross families (RILs crossed to Col and Ler) using a total of 446 published molecular markers. The performance of RILs and additive and dominant components in the test cross families were used to analyze QTLs for 16 traits, using QTL cartographer and composite interval mapping with 1,000 permutations for each trait. Our data suggest that locus-specific and/or genome-wide differential heterozygosity, including epistasis, plays an important role in the generation of the observed heterosis. Furthermore, the hybrid vigor occurred between two closely related ecotypes, and provides a general mechanism for novel variation generated between genetically similar materials.
doi:10.1038/sj.hdy.6800558
PMCID: PMC2041864  PMID: 15316557
hybrid vigor; heterosis; NCIII design; RILs; Arabidopsis; heterozygosity
14.  Quantitative trait loci analysis for rice seed vigor during the germination stage*  
Seed vigor is an important characteristic of seed quality, and rice cultivars with strong seed vigor are desirable in direct-sowing rice production for optimum stand establishment. In the present study, the quantitative trait loci (QTLs) of three traits for rice seed vigor during the germination stage, including germination rate, final germination percentage, and germination index, were investigated using one recombinant inbred line (RIL) population derived from a cross between japonica Daguandao and indica IR28, and using the multiple interval mapping (MIM) approach. The results show that indica rice presented stronger seed vigor during the germination stage than japonica rice. A total of ten QTLs, and at least five novel alleles, were detected to control rice seed vigor, and the amount of variation (R 2) explained by an individual QTL ranged from 7.5% to 68.5%, with three major QTLs with R 2>20%. Most of the QTLs detected here are likely to coincide with QTLs for seed weight, seed size, or seed dormancy, suggesting that the rice seed vigor might be correlated with seed weight, seed size, and seed dormancy. At least five QTLs are novel alleles with no previous reports of seed vigor genes in rice, and those major or minor QTLs could be used to significantly improve the seed vigor by marker-assisted selection (MAS) in rice.
doi:10.1631/jzus.B1000238
PMCID: PMC2997405  PMID: 21121075
Rice; Recombinant inbred line (RIL) population; Seed vigor; Quantitative trait locus (QTL); Germination
15.  Targeted resequencing of a genomic region influencing tameness and aggression reveals multiple signals of positive selection 
Heredity  2011;107(3):205-214.
The identification of the causative genetic variants in quantitative trait loci (QTL) influencing phenotypic traits is challenging, especially in crosses between outbred strains. We have previously identified several QTL influencing tameness and aggression in a cross between two lines of wild-derived, outbred rats (Rattus norvegicus) selected for their behavior towards humans. Here, we use targeted sequence capture and massively parallel sequencing of all genes in the strongest QTL in the founder animals of the cross. We identify many novel sequence variants, several of which are potentially functionally relevant. The QTL contains several regions where either the tame or the aggressive founders contain no sequence variation, and two regions where alternative haplotypes are fixed between the founders. A re-analysis of the QTL signal showed that the causative site is likely to be fixed among the tame founder animals, but that several causative alleles may segregate among the aggressive founder animals. Using a formal test for the detection of positive selection, we find 10 putative positively selected regions, some of which are close to genes known to influence behavior. Together, these results show that the QTL is probably not caused by a single selected site, but may instead represent the joint effects of several sites that were targets of polygenic selection.
doi:10.1038/hdy.2011.4
PMCID: PMC3183948  PMID: 21304545
high-throughput sequencing; sequence capture; positive selection; behavior; QTL mapping
16.  Mapping 49 quantitative trait loci at high resolution through sequencing-based genotyping of rice recombinant inbred lines 
Mapping chromosome regions responsible for quantitative phenotypic variation in recombinant populations provides an effective means to characterize the genetic basis of complex traits. We conducted a quantitative trait loci (QTL) analysis of 150 rice recombinant inbred lines (RILs) derived from a cross between two cultivars, Oryza sativa ssp. indica cv. 93-11 and Oryza sativa ssp. japonica cv. Nipponbare. The RILs were genotyped through next-generation sequencing, which accurately determined the recombination breakpoints and provided a new type of genetic markers, recombination bins, for QTL analysis. We detected 49 QTL with phenotypic effect ranging from 3.2 to 46.0% for 14 agronomics traits. Five QTL of relatively large effect (14.6–46.0%) were located on small genomic regions, where strong candidate genes were found. The analysis using sequencing-based genotyping thus offers a powerful solution to map QTL with high resolution. Moreover, the RILs developed in this study serve as an excellent system for mapping and studying genetic basis of agricultural and biological traits of rice.
Electronic supplementary material
The online version of this article (doi:10.1007/s00122-010-1449-8) contains supplementary material, which is available to authorized users.
doi:10.1007/s00122-010-1449-8
PMCID: PMC3021254  PMID: 20878143
17.  Genetics of Local Adaptation in the Laboratory: Flowering Time Quantitative Trait Loci under Geographic and Seasonal Conditions in Arabidopsis 
PLoS ONE  2006;1(1):e105.
Flowering time in Arabidopsis thaliana is controlled by a large number of genes and various environmental factors, such as light and temperature. The objective of this study was to identify flowering time quantitative trait loci (QTL) under growth conditions simulating seasonal conditions from native geographic locations. Our growth chambers were set to simulate the spring conditions in Spain and Sweden, with appropriate changes in light color, intensity and day length, as well as temperature and relative humidity. Thus the Sweden-like spring conditions changed more dramatically compared to Spain-like spring conditions across the duration of our experiment. We have used these conditions to map QTL responsible for flowering time in the Kas-1/Col-gl1 recombinant inbred lines (RILs) across two replicate blocks. A linkage map from 96 RILs was established using 119 markers including 64 new SNPs markers. One major QTL, mapping to the FRIGIDA (FRI) locus, was detected on the top of chromosome 4 that showed significant gene×seasonal environment interactions. Three other minor QTL also were detected. One QTL mapping near FLOWERING LOCUS M (FLM) showed an epistatic interaction with the QTL at FRI. These QTL×environment and QTL×QTL interactions suggest that subtle ecologically relevant changes in light, temperature, and relative humidity are differentially felt by alleles controlling flowering time and may be responsible for adaptation to regional environments.
doi:10.1371/journal.pone.0000105
PMCID: PMC1762425  PMID: 17205109
18.  Failure to Replicate Two Mate Preference QTLs across Multiple Strains of Drosophila pseudoobscura 
Journal of Heredity  2008;99(6):653-656.
Behavioral genetic mapping studies in model organisms predominantly use crosses originating from a single pair of inbred lines to determine the location of alleles that confer genetic variation in the trait of interest, and they often make sweeping generalizations about the genetic architecture of the trait based on these results. A previous study fine mapped mate preference variation between one pair of Drosophila pseudoobscura lines and identified 2 strong-effect behavioral quantitative trait loci (QTLs). Here, we replicated the previous study's mapping design to examine the extent of variation at these behavioral QTLs across 6 pairs of lines, but we were unable to detect effects of either QTL region in the pairs of lines studied. We suggest that the low-discrimination alleles at these 2 QTLs may occur at low frequency within D. pseudoobscura, although other explanations for the inconsistency are possible. These results underscore the need to examine multiple strains across a species when describing the genetic variation underlying behavioral traits.
doi:10.1093/jhered/esn069
PMCID: PMC2574947  PMID: 18728083
Drosophila; QTL mapping; sexual isolation; species discrimination
19.  Gene Transposition Causing Natural Variation for Growth in Arabidopsis thaliana 
PLoS Genetics  2010;6(5):e1000945.
A major challenge in biology is to identify molecular polymorphisms responsible for variation in complex traits of evolutionary and agricultural interest. Using the advantages of Arabidopsis thaliana as a model species, we sought to identify new genes and genetic mechanisms underlying natural variation for shoot growth using quantitative genetic strategies. More quantitative trait loci (QTL) still need be resolved to draw a general picture as to how and where in the pathways adaptation is shaping natural variation and the type of molecular variation involved. Phenotypic variation for shoot growth in the Bur-0 × Col-0 recombinant inbred line set was decomposed into several QTLs. Nearly-isogenic lines generated from the residual heterozygosity segregating among lines revealed an even more complex picture, with major variation controlled by opposite linked loci and masked by the segregation bias due to the defective phenotype of SG3 (Shoot Growth-3), as well as epistasis with SG3i (SG3-interactor). Using principally a fine-mapping strategy, we have identified the underlying gene causing phenotypic variation at SG3: At4g30720 codes for a new chloroplast-located protein essential to ensure a correct electron flow through the photosynthetic chain and, hence, photosynthesis efficiency and normal growth. The SG3/SG3i interaction is the result of a structural polymorphism originating from the duplication of the gene followed by divergent paralogue's loss between parental accessions. Species-wide, our results illustrate the very dynamic rate of duplication/transposition, even over short periods of time, resulting in several divergent—but still functional—combinations of alleles fixed in different backgrounds. In predominantly selfing species like Arabidopsis, this variation remains hidden in wild populations but is potentially revealed when divergent individuals outcross. This work highlights the need for improved tools and algorithms to resolve structural variation polymorphisms using high-throughput sequencing, because it remains challenging to distinguish allelic from paralogous variation at this scale.
Author Summary
Plant growth is a very complex character impacted by almost any aspect of plant biology and showing continuous variation among natural populations of a single species like Arabidopsis thaliana. Although difficult, it is important to reveal the precise genetic architecture of such a trait's variation to improve our understanding of the mechanisms and evolutionary significance of phenotypic variation. By using recombinant inbred lines derived from a cross between the reference strain ‘Col-0’ and the Irish strain ‘Bur-0’, we have localized several regions of the genome impacting plant growth. When attempting to confirm one of this region's effect, we revealed an even more complex genetic architecture where a first locus (which had remained undetected initially) has a major effect on growth only when a specific genotype was present at a second locus. We have shown here that the reason for this epistatic interaction between the two loci is that the functional allele for a gene important for photosynthesis efficiency and, consequently, growth, had been transposed from one locus to the other in Bur-0 compared to Col-0. This type of structural polymorphism seems to be frequent among strains and, although more difficult to detect, is likely to be of significant evolutionary importance.
doi:10.1371/journal.pgen.1000945
PMCID: PMC2869320  PMID: 20485571
20.  QTL analysis of root traits as related to phosphorus efficiency in soybean 
Annals of Botany  2010;106(1):223-234.
Background and Aims
Low phosphorus (P) availability is a major constraint to soybean growth and production, especially in tropical and subtropical areas. Root traits have been shown to play critical roles in P efficiency in crops. Identification of the quantitative trait loci (QTLs) conferring superior root systems could significantly enhance genetic improvement in soybean P efficiency.
Methods
A population of 106 F9 recombinant inbred lines (RILs) derived from a cross between BD2 and BX10, which contrast in both P efficiency and root architecture, was used for mapping and QTL analysis. Twelve traits were examined in acid soils. A linkage map was constructed using 296 simple sequence repeat (SSR) markers with the Kosambi function, and the QTLs associated with these traits were detected by composite interval mapping and multiple-QTL mapping.
Key Results
The first soybean genetic map based on field data from parental genotypes contrasting both in P efficiency and root architecture was constructed. Thirty-one putative QTLs were detected on five linkage groups, with corresponding contribution ratios of 9·1–31·1 %. Thirteen putative QTLs were found for root traits, five for P content, five for biomass and five for yield traits. Three clusters of QTLs associated with the traits for root and P efficiency at low P were located on the B1 linkage group close to SSR markers Satt519 and Satt519-Sat_128, and on the D2 group close to Satt458; and one cluster was on the B1 linkage group close to Satt519 at high P.
Conclusions
Most root traits in soybean were conditioned by more than two minor QTLs. The region closer to Satt519 on the B1 linkage group might have great potential for future genetic improvement for soybean P efficiency through root selection.
doi:10.1093/aob/mcq097
PMCID: PMC2889805  PMID: 20472699
Quantitative trait loci (QTLs); soybean; Glycine max; root traits; phosphorus efficiency
21.  Quantitative Trait Loci Mapping in Brassica rapa Revealed the Structural and Functional Conservation of Genetic Loci Governing Morphological and Yield Component Traits in the A, B, and C Subgenomes of Brassica Species 
Brassica rapa is an important crop species that produces vegetables, oilseed, and fodder. Although many studies reported quantitative trait loci (QTL) mapping, the genes governing most of its economically important traits are still unknown. In this study, we report QTL mapping for morphological and yield component traits in B. rapa and comparative map alignment between B. rapa, B. napus, B. juncea, and Arabidopsis thaliana to identify candidate genes and conserved QTL blocks between them. A total of 95 QTL were identified in different crucifer blocks of the B. rapa genome. Through synteny analysis with A. thaliana, B. rapa candidate genes and intronic and exonic single nucleotide polymorphisms in the parental lines were detected from whole genome resequenced data, a few of which were validated by mapping them to the QTL regions. Semi-quantitative reverse transcriptase PCR analysis showed differences in the expression levels of a few genes in parental lines. Comparative mapping identified five key major evolutionarily conserved crucifer blocks (R, J, F, E, and W) harbouring QTL for morphological and yield components traits between the A, B, and C subgenomes of B. rapa, B. juncea, and B. napus. The information of the identified candidate genes could be used for breeding B. rapa and other related Brassica species.
doi:10.1093/dnares/dss029
PMCID: PMC3576654  PMID: 23223793
Brassica rapa; quantitative trait loci (QTL); morphological traits; single nucleotide polymorphism (SNP); conserved genome blocks
22.  PHYTOCHROME B and HISTONE DEACETYLASE 6 Control Light-Induced Chromatin Compaction in Arabidopsis thaliana 
PLoS Genetics  2009;5(9):e1000638.
Natural genetic variation in Arabidopsis thaliana exists for many traits and often reflects acclimation to local environments. Studying natural variation has proven valuable in the characterization of phenotypic traits and, in particular, in identifying genetic factors controlling these traits. It has been previously shown that chromatin compaction changes during development and biotic stress. To gain more insight into the genetic control of chromatin compaction, we investigated the nuclear phenotype of 21 selected Arabidopsis accessions from different geographic origins and habitats. We show natural variation in chromatin compaction and demonstrate a positive correlation with latitude of geographic origin. The level of compaction appeared to be dependent on light intensity. A novel approach, combining Quantitative Trait Locus (QTL) mapping and microscopic examination, pointed at PHYTOCHROME-B (PHYB) and HISTONE DEACETYLASE-6 (HDA6) as positive regulators of light-controlled chromatin compaction. Indeed, mutant analyses demonstrate that both factors affect global chromatin organization. HDA6, in addition, strongly promotes the light-mediated compaction of the Nucleolar Organizing Regions (NORs). The accession Cape Verde Islands-0 (Cvi-0), which shows sequence polymorphism in the PHYB gene and in the HDA6 promotor, resembles the hda6 mutant in having reduced chromatin compaction and decreased methylation levels of DNA and histone H3K9 at the NORs. We provide evidence that chromatin organization is controlled by light intensity. We propose that chromatin plasticity is associated with acclimation of Arabidopsis to its environment. The polymorphic alleles such as PHYB and HDA6 control this process.
Author Summary
The habitat of the plant model species Arabidopsis thaliana can be found throughout the Northern hemisphere. As a consequence, individual populations have acclimated to a great diversity of environmental conditions. This is reflected by a wealth of natural genetic variation in many phenotypic traits. We utilized this natural variation via a novel approach, combining microscopic examination, quantitative genetics, and analysis of environmental parameters, to understand the regulation of nuclear chromatin compaction in leaf mesophyll cells. We show that the level of chromatin compaction among natural Arabidopsis thaliana accessions correlates with latitude of origin and depends on local light intensity. Our study provides evidence that the photoreceptor PHYTOCHROME-B (PHYB) and the histone modifier HISTONE DEACETYLASE 6 (HDA6) are positive regulators of global chromatin organization in a light-dependent manner. In addition, HDA6 specifically controls light-mediated chromatin compaction of the Nucleolar Organizing Regions (NORs). We propose that the observed light-controlled plasticity of chromatin plays a role in acclimation and survival of plants in their natural environment.
doi:10.1371/journal.pgen.1000638
PMCID: PMC2728481  PMID: 19730687
23.  Mapping of QTL for Resistance against the Crucifer Specialist Herbivore Pieris brassicae in a New Arabidopsis Inbred Line Population, Da(1)-12×Ei-2 
PLoS ONE  2007;2(6):e578.
Background
In Arabidopsis thaliana and other crucifers, the glucosinolate-myrosinase system contributes to resistance against herbivory by generalist insects. As yet, it is unclear how crucifers defend themselves against crucifer-specialist insect herbivores.
Methodology/Principal Findings
We analyzed natural variation for resistance against two crucifer specialist lepidopteran herbivores, Pieris brassicae and Plutella xylostella, among Arabidopsis thaliana accessions and in a new Arabidopsis recombinant inbred line (RIL) population generated from the parental accessions Da(1)-12 and Ei-2. This RIL population consists of 201 individual F8 lines genotyped with 84 PCR-based markers. We identified six QTL for resistance against Pieris herbivory, but found only one weak QTL for Plutella resistance. To elucidate potential factors causing these resistance QTL, we investigated leaf hair (trichome) density, glucosinolates and myrosinase activity, traits known to influence herbivory by generalist insects. We identified several previously unknown QTL for these traits, some of which display a complex pattern of epistatic interactions.
Conclusions/Significance
Although some trichome, glucosinolate or myrosinase QTL co-localize with Pieris QTL, none of these traits explained the resistance QTL convincingly, indicating that resistance against specialist insect herbivores is influenced by other traits than resistance against generalists.
doi:10.1371/journal.pone.0000578
PMCID: PMC1892800  PMID: 17593977
24.  QTL mapping of agronomic traits in tef [Eragrostis tef (Zucc) Trotter] 
BMC Plant Biology  2007;7:30.
Background
Tef [Eragrostis tef (Zucc.) Trotter] is the major cereal crop in Ethiopia. Tef is an allotetraploid with a base chromosome number of 10 (2n = 4× = 40) and a genome size of 730 Mbp. The goal of this study was to identify agronomically important quantitative trait loci (QTL) using recombinant inbred lines (RIL) derived from an inter-specific cross between E. tef and E. pilosa (30-5).
Results
Twenty-two yield-related and morphological traits were assessed across eight different locations in Ethiopia during the growing seasons of 1999 and 2000. Using composite interval mapping and a linkage map incorporating 192 loci, 99 QTLs were identified on 15 of the 21 linkage groups for 19 traits. Twelve QTLs on nine linkage groups were identified for grain yield. Clusters of more than five QTLs for various traits were identified on seven linkage groups. The largest cluster (10 QTLs) was identified on linkage group 8; eight of these QTLs were for yield or yield components, suggesting linkage or pleotrophic effects of loci. There were 15 two-way interactions of loci to detect potential epistasis identified and 75% of the interactions were derived from yield and shoot biomass. Thirty-one percent of the QTLs were observed in multiple environments; two yield QTLs were consistent across all agro-ecology zones. For 29.3% of the QTLs, the alleles from E. pilosa (30-5) had a beneficial effect.
Conclusion
The extensive QTL data generated for tef in this study will provide a basis for initiating molecular breeding to improve agronomic traits in this staple food crop for the people of Ethiopia.
doi:10.1186/1471-2229-7-30
PMCID: PMC1913516  PMID: 17565675
25.  Genetic mapping of natural variation in a shade avoidance response: ELF3 is the candidate gene for a QTL in hypocotyl growth regulation 
Journal of Experimental Botany  2010;62(1):167-176.
When plants become shaded by neighbouring plants, they perceive a decrease in the red/far-red (R/FR) ratio of the light environment, which provides an early and unambiguous warning of the presence of competing vegetation. The mechanistic bases of the natural genetic variation in response to shade signals remain largely unknown. This study demonstrates that a wide range of genetic variation for hypocotyl elongation in response to an FR pulse at the end of day (EOD), a light signal that simulates natural shade, exists between Arabidopsis accessions. A quantitative trait locus (QTL) mapping analysis was done in the Bayreuth×Shahdara recombinant inbred line population. EODINDEX1 is the most significant QTL identified in response to EOD. The Shahdara alleles at EODINDEX1 caused a reduced response to shade as a consequence of an impaired hypocotyl inhibition under white light, and an accelerated leaf movement rhythm, which correlated positively with the pattern of circadian expression of clock genes such as PRR7 and PRR9. Genetic and quantitative complementation analyses demonstrated that ELF3 is the most likely candidate gene underlying natural variation at EODINDEX1. In conclusion, ELF3 is proposed as a component of the shade avoidance signalling pathway responsible for the phenotypic differences between Arabidopsis populations in relation to adaptation in a changing light environment.
doi:10.1093/jxb/erq253
PMCID: PMC2993907  PMID: 20713464
Arabidopsis thaliana; early flowering 3 (ELF3); end of the day far-red light (EOD); natural genetic variation; quantitative trait locus (QTL); shade avoidance syndrome (SAS)

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