Metabolite profiling and quantitative genetics of natural variation for flavonoids in Arabidopsis

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.     

Quantitative trait loci controlling root growth and architecture in Arabidopsis thaliana confirmed by heterogeneous inbred family

Arabidopsis thaliana provides a scientifically attractive and simple model for studying root growth and architecture and, subsequently, for discovering new genes involved in the control of these characters in plants. We have used the natural variation available in Arabidopsis accessions and mapped quantitative trait loci (QTLs) for primary root length (PRL), lateral root number (LRN) and density (LRD) and for total length of the lateral root system (LRL) in the Bay-0 × Shahdara population. Total phenotypic variation was very large, and despite the importance of the environmental component we were able to map 13 QTLs and one epistatic interaction between QTLs. Our results highlight the biological relevance and genetic control of lateral root density in this material. We were also able to show that variation in the extent of the lateral root system depends mainly on the growth of the existing lateral roots rather than in a change in LRN. Factors controlling lateral root growth seemed to have no major effect on primary root growth. Moreover, Shahdara QTL alleles always increased the length of the lateral roots, which may be taken as an adaptation to its very dry natural environment in Tadjikistan. A QTL for PRL was confirmed using a type of near-isogenic line called a heterogeneous inbred family (HIF), and this QTL is a candidate for further fine-mapping and cloning.     

Natural variation for carbohydrate content in Arabidopsis. Interaction with complex traits dissected by quantitative genetics

Besides being a metabolic fuel, carbohydrates play important roles in plant growth and development, in stress responses, and as signal molecules. We exploited natural variation in Arabidopsis (Arabidopsis thaliana) to decipher the genetic architecture determining carbohydrate content. A quantitative trait locus (QTL) approach in the Bay-0 x Shahdara progeny grown in two contrasting nitrogen environments led to the identification of 39 QTLs for starch, glucose, fructose, and sucrose contents representing at least 14 distinct polymorphic loci. A major QTL for fructose content (FR3.4) and a QTL for starch content (ST3.4) were confirmed in heterogeneous inbred families. Several genes associated with carbon (C) metabolism colocalize with the identified QTL. QTLs for senescence-related traits, and for flowering time, water status, and nitrogen-related traits, previously detected with the same genetic material, colocalize with C-related QTLs. These colocalizations reflect the complex interactions of C metabolism with other physiological processes. QTL fine-mapping and cloning could thus lead soon to the identification of genes potentially involved in the control of different connected physiological processes.     

Quantitative trait loci analysis of water and anion contents in interaction with nitrogen availability in Arabidopsis thaliana

In plants, water and anion parameters are linked, for example through the integration of nutritional signaling and the response to diverse stress. In this work, Arabidopsis thaliana is used as a model system to dissect the genetic variation of these parameters by quantitative trait loci (QTL) mapping in the 415 recombinant inbred lines of the Bay-0 x Shahdara population. Water, nitrate, chloride, and phosphate contents were measured at the vegetative stage in the shoots of plants grown in controlled conditions. Two contrasting nitrogen (N) conditions were studied, one leading to the complete depletion of the nitrate pool in the plants. Most of the observed genetic variation was identified as QTL, with medium but also large phenotypic contributions. QTL colocalization provides a genetic basis for the correlation between water and nitrate contents in nonlimiting N conditions and water and chloride contents in limiting N conditions. The 34 new QTL described here represent at least 19 loci polymorphic between Bay-0 and Shahdara; some may correspond to known genes from water/anion transport systems, while others clearly identify new genes controlling or interacting with water/anion absorption and accumulation. Interestingly, flowering-time genes probably play a role in the regulation of water content in our conditions.     

拟南芥低温黑暗发芽基因的鉴定及QTL定位

低温发芽是一个重要农艺性状,提高作物的耐寒性并确定控制低温发芽的基因是一个全球性的重大育种课题。通过对300多份拟南芥种子进行发芽试验,鉴定了低温黑暗下发芽的自然变异,并比较了两种生态型Bay-0和Shahdara低温黑暗下的发芽特性,这种自然变异被用来鉴定低温黑暗中发芽基因的遗传位点;利用这两个生态型杂交产生的重组近交系,结合分子标记和QTL方法,检测到种子在低温黑暗下发芽至少受6个QTL所控制,其中3个为主效位点,它们所解释的自然变异为61%,利用异源近亲代(HIF)验证了这3个主要QTL,为低温黑暗发芽基因的精细定位和基因克隆等研究奠定了基础。     

Natural Variation in Seed Very Long Chain Fatty Acid Content Is Controlled by a New Isoform of KCS18 in Arabidopsis thaliana

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.     

Network Analysis Identifies ELF3 as a QTL for the Shade Avoidance Response in Arabidopsis

Quantitative Trait Loci (QTL) analyses in immortal populations are a powerful method for exploring the genetic mechanisms that control interactions of organisms with their environment. However, QTL analyses frequently do not culminate in the identification of a causal gene due to the large chromosomal regions often underlying QTLs. A reasonable approach to inform the process of causal gene identification is to incorporate additional genome-wide information, which is becoming increasingly accessible. In this work, we perform QTL analysis of the shade avoidance response in the Bayreuth-0 (Bay-0, CS954) x Shahdara (Sha, CS929) recombinant inbred line population of Arabidopsis. We take advantage of the complex pleiotropic nature of this trait to perform network analysis using co-expression, eQTL and functional classification from publicly available datasets to help us find good candidate genes for our strongest QTL, SAR2. This novel network analysis detected EARLY FLOWERING 3 (ELF3; AT2G25930) as the most likely candidate gene affecting the shade avoidance response in our population. Further genetic and transgenic experiments confirmed ELF3 as the causative gene for SAR2. The Bay-0 and Sha alleles of ELF3 differentially regulate developmental time and circadian clock period length in Arabidopsis, and the extent of this regulation is dependent on the light environment. This is the first time that ELF3 has been implicated in the shade avoidance response and that different natural alleles of this gene are shown to have phenotypic effects. In summary, we show that development of networks to inform candidate gene identification for QTLs is a promising technique that can significantly accelerate the process of QTL cloning.     

Quantitative trait loci analysis of nitrogen use efficiency in Arabidopsis

Improving plant nitrogen (N) use efficiency or controlling soil N requires a better knowledge of the regulation of plant N metabolism. This could be achieved using Arabidopsis as a model genetic system, taking advantage of the natural variation available among ecotypes. Here, we describe an extensive study of N metabolism variation in the Bay-0 x Shahdara recombinant inbred line population, using quantitative trait locus (QTL) mapping. We mapped QTL for traits such as shoot growth, total N, nitrate, and free-amino acid contents, measured in two contrasting N environments (contrasting nitrate availability in the soil), in controlled conditions. Genetic variation and transgression were observed for all traits, and most of the genetic variation was identified through QTL and QTL x QTL epistatic interactions. The 48 significant QTL represent at least 18 loci that are polymorphic between parents; some may correspond to known genes from the N metabolic pathway, but others represent new genes controlling or interacting with N physiology. The correlations between traits are dissected through QTL colocalizations: The identification of the individual factors contributing to the regulation of different traits sheds new light on the relations among these characters. We also point out that the regulation of our traits is mostly specific to the N environment (N availability). Finally, we describe four interesting loci at which positional cloning is feasible.     

Genetic analysis of the physiological responses to low boron stress in Arabidopsis thaliana

Boron (B) is an essential micronutrient for higher plants. There is wide genetic variation in the response to B deficiency among plant species and cultivars. The objective of this study was to identify quantitative trait loci (QTL) that control B efficiency in natural Arabidopsis accessions. The B efficiency coefficient (BEC) and seed yield under low B conditions (SYLB) were investigated by solution culture in two separate experiments in an Arabidopsis recombinant inbred line (RIL) population. Both of the traits studied exhibited high transgressive variation in the RIL population, and, in total, five and three QTL were identified for BEC and SYLB, respectively. Three of the five QTL, including the QTL, AtBE1-2, that has a large effect on the BEC, were found at the interval of the corresponding QTL for SYLB in both experiments. The close genetic relationship between BEC and SYLB was further confirmed by conditional QTL mapping in the RIL population and unconditional QTL mapping in an AtBE1-2-segregated F(2) population. Epistatic interactions for the tested traits were analysed, and were found to be widespread in the detected QTL of Arabidopsis in the RIL population. Comparison of the QTL interval for B efficiency with reported B-related genes showed that 10 B-related genes, together with one BOR1 homolog (BOR5, At1g74810) were located in the QTL region of AtBE1-2. These results suggest that natural variation in B efficiency in Arabidopsis has a complex molecular basis. They also provide a basis for fine mapping and cloning of the B-efficiency genes, with the ultimate aim of discovering the physiological mechanism of action of the genes.     

Lz-0 × Berkeley: a new Arabidopsis recombinant inbred line population for the mapping of complex traits

This study describes the generation and test of a genetic resource suited to identify determinants of cell biological traits in plants. The use of quantitative trait loci (QTL) mapping for a better genetic understanding of cell biological traits is still at an early stage, even for biotechnologically important cell properties, such as the dimensions of fiber cells. A common strategy, the mapping of QTLs in recombinant inbred line (RIL) populations, is limited by the fact that the existing RIL populations exploit only a small fraction of the existing natural variation. Here, we report the mapping of QTLs impacting on the length of fiber cells in Arabidopsis inflorescence stems in a newly generated RIL population derived from a cross between the accessions Berkeley and the little known Lz-0. Through inbreeding of individual F₂ plants, a total of 159 new F₈ lines were produced and genotyped with a set of 49 single nucleotide polymorphism markers. The population was successfully used not only for the mapping of three QTLs controlling fiber length, but also to map five QTL controlling flowering time under short and long-day conditions. Our study demonstrates the usefulness of this new genetic resource by mapping in it QTLs underlying a poorly explored cellular trait as well as an already better explored regulatory pathway. The new RIL population and an online platform for the continuous supplementation of genetic markers will be generally available to substantially broaden the genetic diversity through which loci with impact on plant quantitative traits can be identified.     

Leaf yellowing and anthocyanin accumulation are two genetically independent strategies in response to nitrogen limitation in Arabidopsis thaliana

For the first time in Arabidopsis thaliana, this work proposes the identification of quantitative trait loci (QTLs) associated with leaf senescence and stress response symptoms such as yellowing and anthocyanin-associated redness. When Arabidopsis plants were cultivated under low nitrogen conditions, we observed that both yellowing of the old leaves of the rosette and whole rosette redness were promoted. Leaf yellowing is a senescence symptom related to chlorophyll breakdown. Redness is a symptom of anthocyanin accumulation related to whole plant ageing and nutrient limitation. In this work, Arabidopsis is used as a model system to dissect the genetic variation of these parameters by QTL mapping in the 415 recombinant inbred lines of the Bay-0xShahdara population. Fifteen new QTLs and two epistatic interactions were described in this study. The yellowing of the rosette, estimated by visual notation and image processing, was controlled by four and five QTLs, respectively. The visual estimation of redness allowed us to detect six QTLs among which the major one explained 33% of the total variation. Two main QTLs were confirmed in near-isogenic lines (heterogenous inbred family; HIF), thus confirming the relevance of the visual notation of these traits. Co-localizations between QTLs for leaf yellowing, redness and nitrogen use efficiency described in a previous publication indicate complex interconnected pathways involved in both nitrogen management and senescence- and stress-related processes. No co-localization between QTLs for leaf yellowing and redness has been found, suggesting that the two characters are genetically independent.     

Defining the genetic architecture underlying female- and male-mediated nonrandom mating and seed yield traits in Arabidopsis

Postpollination nonrandom mating among compatible mates is a widespread phenomenon in plants and is genetically undefined. In this study, we used the recombinant inbred line (RIL) population between Landsberg erecta and Columbia (Col) accessions of Arabidopsis (Arabidopsis thaliana) to define the genetic architecture underlying both female- and male-mediated nonrandom mating traits. To map the genetic loci responsible for male-mediated nonrandom mating, we performed mixed pollinations with Col and RIL pollen on Col pistils. To map the genetic loci responsible for female-mediated nonrandom mating, we performed mixed pollinations with Col and Landsberg erecta pollen on RIL pistils. With these data, we performed composite interval mapping to identify two quantitative trait loci (QTLs) that control male-mediated nonrandom mating. We detected epistatic interactions between these two loci. We also explored female- and male-mediated traits involved in seed yield in mixed pollinations. We detected three female QTLs and one male QTL involved in directing seed number per fruit. To our knowledge, the results of these experiments represent the first time the female and male components of seed yield and nonrandom mating have been separately mapped.     

Genetic variation at bx1 controls DIMBOA content in maize

The main hydroxamic acid in maize (Zea mays L.) is 2-4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA). DIMBOA confers resistance to leaf-feeding by several corn borers. Most genes involved in the DIMBOA metabolic pathway are located on the short arm of chromosome 4, and quantitative trait loci (QTLs) involved in maize resistance to leaf-feeding by corn borers have been localized to that region. However, the low resolution of QTL linkage mapping does not allow convincing proof that genetic variation at bx loci was responsible for the variability for resistance. This study addressed the following objectives: to determine the QTLs involved in DIMBOA synthesis across genetically divergent maize inbreds using eight RIL families from the nested association mapping population, to check the stability of QTLs for DIMBOA content across years by evaluating two of those RIL families in 2 years, and to test the involvement of bx1 by performing association mapping with a panel of 281 diverse inbred lines. QTLs were stable across different environments. A genetic model including eight markers explained approximately 34% of phenotypic variability across eight RIL families and the position of the largest QTL co-localizes with the majority of structural genes of the DIMBOA pathway. Candidate association analysis determined that sequence polymorphisms at bx1 greatly affects variation of DIMBOA content in a diverse panel of maize inbreds, but the specific causal polymorphism or polymorphisms responsible for the QTL detected in the region 4.01 were not identified. This result may be because the causal polymorphism(s) were not sequenced, identity is masked by linkage disequilibrium, adjustments for population structure reduce significance of causal polymorphisms or multiple causal polymorphisms affecting bx1 segregate among inbred lines.     

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.     

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%.     

Variation in seed dormancy quantitative trait loci in Arabidopsis thaliana originating from one site

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.     

Genetic Architecture of Natural Variation of Telomere Length in Arabidopsis thaliana

Telomeres represent the repetitive sequences that cap chromosome ends and are essential for their protection. Telomere length is known to be highly heritable and is derived from a homeostatic balance between telomeric lengthening and shortening activities. Specific loci that form the genetic framework underlying telomere length homeostasis, however, are not well understood. To investigate the extent of natural variation of telomere length in Arabidopsis thaliana, we examined 229 worldwide accessions by terminal restriction fragment analysis. The results showed a wide range of telomere lengths that are specific to individual accessions. To identify loci that are responsible for this variation, we adopted a quantitative trait loci (QTL) mapping approach with multiple recombinant inbred line (RIL) populations. A doubled haploid RIL population was first produced using centromere-mediated genome elimination between accessions with long (Pro-0) and intermediate (Col-0) telomere lengths. Composite interval mapping analysis of this population along with two established RIL populations (Ler-2/Cvi-0 and Est-1/Col-0) revealed a number of shared and unique QTL. QTL detected in the Ler-2/Cvi-0 population were examined using near isogenic lines that confirmed causative regions on chromosomes 1 and 2. In conclusion, this work describes the extent of natural variation of telomere length in A. thaliana, identifies a network of QTL that influence telomere length homeostasis, examines telomere length dynamics in plants with hybrid backgrounds, and shows the effects of two identified regions on telomere length regulation.     

Identification of QTLs for eight agronomically important traits using an ultra-high-density map based on SNPs generated from high-throughput sequencing in sorghum under contrasting photoperiods

The productivity of sorghum is mainly determined by agronomically important traits. The genetic bases of these traits have historically been dissected and analysed through quantitative trait locus (QTL) mapping based on linkage maps with low-throughput molecular markers, which is one of the factors that hinder precise and complete information about the numbers and locations of the genes or QTLs controlling the traits. In this study, an ultra-high-density linkage map based on high-quality single nucleotide polymorphisms (SNPs) generated from low-coverage sequences (~0.07 genome sequence) in a sorghum recombinant inbred line (RIL) population was constructed through new sequencing technology. This map consisted of 3418 bin markers and spanned 1591.4 cM of genome size with an average distance of 0.5 cM between adjacent bins. QTL analysis was performed and a total of 57 major QTLs were detected for eight agronomically important traits under two contrasting photoperiods. The phenotypic variation explained by individual QTLs varied from 3.40% to 33.82%. The high accuracy and quality of this map was evidenced by the finding that genes underlying two cloned QTLs, Dw3 for plant height (chromosome 7) and Ma1 for flowering time (chromosome 6), were localized to the correct genomic regions. The close associations between two genomic regions on chromosomes 6 and 7 with multiple traits suggested the existence of pleiotropy or tight linkage. Several major QTLs for heading date, plant height, numbers of nodes, stem diameter, panicle neck length, and flag leaf width were detected consistently under both photoperiods, providing useful information for understanding the genetic mechanisms of the agronomically important traits responsible for the change of photoperiod.