High-throughput root phenotyping screens identify genetic loci associated with root architectural traits in Brassica napus under contrasting phosphate availabilities

Background and Aims

Phosphate (Pi) deficiency in soils is a major limiting factor for crop growth worldwide. Plant growth under low Pi conditions correlates with root architectural traits and it may therefore be possible to select these traits for crop improvement. The aim of this study was to characterize root architectural traits, and to test quantitative trait loci (QTL) associated with these traits, under low Pi (LP) and high Pi (HP) availability in Brassica napus.

Methods

Root architectural traits were characterized in seedlings of a double haploid (DH) mapping population (n = 190) of B. napus [‘Tapidor’ × ‘Ningyou 7’ (TNDH)] using high-throughput phenotyping methods. Primary root length (PRL), lateral root length (LRL), lateral root number (LRN), lateral root density (LRD) and biomass traits were measured 12 d post-germination in agar at LP and HP.

Key Results

In general, root and biomass traits were highly correlated under LP and HP conditions. ‘Ningyou 7’ had greater LRL, LRN and LRD than ‘Tapidor’, at both LP and HP availability, but smaller PRL. A cluster of highly significant QTL for LRN, LRD and biomass traits at LP availability were identified on chromosome A03; QTL for PRL were identified on chromosomes A07 and C06.

Conclusions

High-throughput phenotyping of Brassica can be used to identify root architectural traits which correlate with shoot biomass. It is feasible that these traits could be used in crop improvement strategies. The identification of QTL linked to root traits under LP and HP conditions provides further insights on the genetic basis of plant tolerance to P deficiency, and these QTL warrant further dissection.     

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.     

Mapping QTLs and candidate genes for rice root traits under different water-supply conditions and comparative analysis across three populations

To investigate the genetic factors underlying constitutive and adaptive morphological traits of roots under different water-supply conditions, a recombinant inbred line (RIL) population derived from a cross between the lowland rice variety IR1552 and the upland rice variety Azucena with 249 molecular markers, was used in cylindrical-pot experiments. Eighteen QTLs were detected for seminal root length (SRL), adventitious root number (ARN), and lateral root length (LRL) and lateral root number (LRN) on the seminal root at a soil depth of from 3 to 6 cm under flooding and upland conditions. One identical QTL was detected under both flooding and upland conditions. The relative parameters under the two water-supply conditions were also used for QTL analysis. Five QTLs for upland induced variations in the traits were detected with the positive alleles from Azucena. A comparative analysis was performed for the QTLs detected in this study and those reported from two other populations with Azucena as a parent. Several identical QTLs for root elongation were found across the three populations with positive alleles from Azucena. Candidate genes were screened from ESTs and cDNA-AFLP clones for comparative mapping with the detected QTLs. Two genes for cell expansion, OsEXP2 and endo-1,4--D-glucanase EGase, and four cDNA-AFLP clones from root tissues of Azucena, were mapped on the intervals carrying the QTLs for SRL and LRL under upland conditions, respectively.Communicated by H.C. Becker     

Mapping QTLs for root traits under different nitrate levels at the seedling stage in maize (Zea mays L.)

Nitrogen (N) loss is a worldwide problem in crop production. Apart from reasonable N fertilizer application, breeding N efficient cultivars provides an alternative way. Root architecture is an important factor determining N acquisition. However, little is known about the molecular genetic basis for root growth in relation to N supply. In the present study, an F₈ maize (Zea may L.) recombinant inbred (RI) population consisting of 94 lines was used to identify the QTLs for root traits under different nitrate levels. The lateral root length (LRL), axial root length (ARL), maximal axial root length (MARL), axial root number (ARN) and average axial root length (AARL) were evaluated under low N (LN) and high N (HN) conditions in a hydroponics system. A total of 17 QTLs were detected among which 14 loci are located on the same chromosome region as published QTLs for root traits. A major QTL on chromosome 1 (between bnlg1025 and umc2029) for the AARL under LN could explain 43.7% of the phenotypic variation. This QTL co-localizes with previously reported QTLs that associate with root traits, grain yield, and N uptake. Our results indicate that longer axial roots are important for efficient N acquisition and the major QTL for AARL may be used as a marker in breeding N efficient maize genotypes.     

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.     

Bay-0 x Shahdara recombinant inbred line population: a powerful tool for the genetic dissection of complex traits in Arabidopsis

Natural genetic variation in Arabidopsis is considerable, but has not yet been used extensively as a source of variants to identify new genes of interest. From the cross between two genetically distant ecotypes, Bay-0 and Shahdara, we generated a Recombinant Inbred Line (RIL) population dedicated to Quantitative Trait Locus (QTL) mapping. A set of 38 physically anchored microsatellite markers was created to construct a robust genetic map from the 420 F6 lines. These markers, evenly distributed throughout the five chromosomes, revealed a remarkable equilibrium in the segregation of parental alleles in the genome. As a model character, we have analysed the genetic basis of variation in flowering time in two different environments. The simultaneous mapping of both large- and small-effect QTLs responsible for this variation explained 90% of the total genotypic variance. Two of the detected QTLs colocalize very precisely with FRIGIDA and FLOWERING LOCUS C genes; we provide information on the polymorphism of genes confirming this hypothesis. Another QTL maps in a region where no QTL had been found previously for this trait. This confirms the accuracy of QTL detection using the Bay-0 x Shahdara RIL population, which constitutes the largest in size available so far in Arabidopsis. As an alternative to mutant analysis, this population represents a powerful tool which is currently being used to undertake the genetic dissection of complex metabolic pathways.     

Major quantitative trait loci for seminal root morphology of wheat seedlings

Vigorous early root growth at seedling stage has been shown to be important for efficient acquisition of nutrients in wheat (Triticum aestivum L.). Identifying quantitative trait loci (QTL) for early root growth can facilitate the selection of wheat varieties with efficient nutrient use. A recombinant inbred line population derived from two Chinese wheat varieties, Xiaoyan 54 and Jing 411, was grown hydroponically at seedling stage. The maximum root length (MRL), primary root length (PRL), lateral root length (LRL), total root length (TRL), and root tip number (RN) of seminal roots were measured using the WinRHIZO Root Analyser. Numerous QTL for the investigated root traits were detected with QTL numbers varying from two to six, depending on the traits. Among them, two loci had major effects on primary (MRL and PRL) and lateral (LRL and RN) root parameters, respectively. The QTL (namely qTaLRO-B1) between Xgwm210 and Xbarc1138.2 on chromosome 2B explained 68.0 and 59.0% of phenotypic variations in MRL and PRL, respectively; the major QTL between Xgwm570 and Xgwm169.2 on chromosome 6A explained 30.5 and 24.5% of phenotypic variations in LRL and RN, respectively. These two major loci showed linkage with previous reported QTL for yield component and nutrient uptake. Detailed analysis of qTaLRO-B1 indicated that the positive allele of qTaLRO-B1 showed dominance over the negative allele, which showed impairment in primary root elongation. The existence of major QTL for root trait and their linkage with agronomic traits and nutrient uptake will facilitate the design of root morphology for better yield performance and efficient nutrient use.     

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.     

QTLs for the elongation of axile and lateral roots of maize in response to low water potential

Changes in root architecture and the maintenance of root growth in drying soil are key traits for the adaptation of maize (Zea mays L.) to drought environments. The goal of this study was to map quantitative trait loci (QTLs) for root growth and its response to dehydration in a population of 208 recombinant inbred lines from the International Maize and Wheat Improvement Center (CIMMYT). The parents, Ac7643 and Ac7729/TZSRW, are known to be drought-tolerant and drought-sensitive, respectively. Roots were grown in pouches under well-watered conditions or at low water potential induced by the osmolyte polyethylene glycol (PEG 8000). Axile root length (L _Ax) increased linearly, while lateral root length (L _Lat) increased exponentially over time. Thirteen QTLs were identified for six seedling traits: elongation rates of axile roots (ER_Ax), the rate constant of lateral root elongation (k _Lat), the final respective lengths (L _Ax and L _Lat), and the ratios k _Lat/ER_Ax and L _Lat/L _Ax. While QTLs for lateral root traits were constitutively expressed, most QTLs for axile root traits responded to water stress. For axile roots, common QTLs existed for ER_Ax and L _Ax. Quantitative trait loci for the elongation rates of axile roots responded more clearly to water stress compared to root length. Two major QTLs were detected: a QTL for general vigor in bin 2.02, affecting most of the traits, and a QTL for the constitutive increase in k _Lat and k _Lat/ER_Ax in bins 6.04–6.05. The latter co-located with a major QTL for the anthesis-silking interval (ASI) reported in published field experiments, suggesting an involvement of root morphology in drought tolerance. Rapid seedling tests are feasible for elucidating the genetic response of root growth to low water potential. Some loci may even have pleiotropic effects on yield-related traits under drought stress.     

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.     

A dissection model for mapping complex traits

Many quantitative traits are composites of other traits that contribute differentially to genetic variation. Quantitative trait locus (QTL) mapping of these composite traits can benefit by incorporating the mechanistic process of how their formation is mediated by the underlying components. We propose a dissection model by which to map these interconnected components traits under a joint likelihood setting. The model can test how a composite trait is determined by pleiotropic QTLs for its component traits or jointly by different sets of QTLs each responsible for a different component. The model can visualize the pattern of time‐varying genetic effects for individual components and their impacts on composite traits. The dissection model was used to map two composite traits, stemwood volume growth decomposed into its stem height, stem diameter and stem form components for Euramerican poplar adult trees, and total lateral root length constituted by its average lateral root length and lateral root number components for Euphrates poplar seedlings. We found the pattern of how QTLs for different components contribute to phenotypic variation in composite traits. The detailed understanding of the genetic machineries of composite traits will not only help in the design of molecular breeding in plants and animals, but also shed light on the evolutionary processes of quantitative traits under natural selection.     

Visualizing the genetic landscape of Arabidopsis seed performance

Perfect timing of germination is required to encounter optimal conditions for plant survival and is the result of a complex interaction between molecular processes, seed characteristics, and environmental cues. To detangle these processes, we made use of natural genetic variation present in an Arabidopsis (Arabidopsis thaliana) Bayreuth × Shahdara recombinant inbred line population. For a detailed analysis of the germination response, we characterized rate, uniformity, and maximum germination and discuss the added value of such precise measurements. The effects of after-ripening, stratification, and controlled deterioration as well as the effects of salt, mannitol, heat, cold, and abscisic acid (ABA) with and without cold stratification were analyzed for these germination characteristics. Seed morphology (size and length) of both dry and imbibed seeds was quantified by using image analysis. For the overwhelming amount of data produced in this study, we developed new approaches to perform and visualize high-throughput quantitative trait locus (QTL) analysis. We show correlation of trait data, (shared) QTL positions, and epistatic interactions. The detection of similar loci for different stresses indicates that, often, the molecular processes regulating environmental responses converge into similar pathways. Seven major QTL hotspots were confirmed using a heterogeneous inbred family approach. QTLs colocating with previously reported QTLs and well-characterized mutants are discussed. A new connection between dormancy, ABA, and a cripple mucilage formation due to a naturally occurring mutation in the MUCILAGE-MODIFIED2 gene is proposed, and this is an interesting lead for further research on the regulatory role of ABA in mucilage production and its multiple effects on germination parameters.     

磷空间有效性对拟南芥根形态构型的影响

磷空间有效性显著影响拟南芥主、侧根生长。在均一的磷处理下,极度磷胁迫或过量供磷均会导致拟南芥主根变短和侧根密度降低。在分层的磷处理下,上层高磷下层低磷能明显促进主根伸长生长,提高侧根在高磷区域的密度,说明植物根系在下层低磷区感受到磷胁迫信号后,可促进上层高磷区侧根的形成和发育。     

Genetic dissection of root growth in rice (Oryza sativa L.). II: mapping quantitative trait loci using molecular markers

Drought is a major abiotic stress of upland rice, and good root growth has been associated with drought avoidance. We report on the genetic mapping of root growth traits in an F₂ population derived from two drought-resistant rice varieties, ‘Bala’ and ‘Azucena’. Restriction fragment length polymorphism (RFLP) between the parents was 32%, and a molecular map with 71 marker loci and 17 linkage groups covering 1280 cM was produced. Quantitative trait loci (QTLs) for eight root growth characteristics were mapped using phenotype data obtained in a hydroponic screen previously described in a companion paper. Using a significance threshold of LOD 2.4, we observed one QTL for maximum root length after 28 days growth on chromosome 11. It had a LOD score of 6.9, explained nearly 30% of the variation and appeared to be largely additive in effect. QTLs for maximum root length after 3, 7, 14 and 21 days of growth were also revealed. Some root-length QTLs, including that on chromosome 11, varied greatly with developmental stage. One QTL for root volume and two QTLs for adventitious root thickness were detected. No QTLs were detected for the length of cells in the mature (fully expanded) zone of adventitious root tips. The results obtained are discussed in the context of previous reports on mapping root growth parameters in rice.     

QTL mapping and phenotypic variation for root architectural traits in maize (Zea mays L.)

Key message

QTL were identified for root architectural traits in maize.

Abstract

Root architectural traits, including the number, length, orientation, and branching of the principal root classes, influence plant function by determining the spatial and temporal domains of soil exploration. To characterize phenotypic patterns and their genetic control, three recombinant inbred populations of maize were grown for 28 days in solid media in a greenhouse and evaluated for 21 root architectural traits, including length, number, diameter, and branching of seminal, primary and nodal roots, dry weight of embryonic and nodal systems, and diameter of the nodal root system. Significant phenotypic variation was observed for all traits. Strong correlations were observed among traits in the same root class, particularly for the length of the main root axis and the length of lateral roots. In a principal component analysis, relationships among traits differed slightly for the three families, though vectors grouped together for traits within a given root class, indicating opportunities for more efficient phenotyping. Allometric analysis showed that trajectories of growth for specific traits differ in the three populations. In total, 15 quantitative trait loci (QTL) were identified. QTL are reported for length in multiple root classes, diameter and number of seminal roots, and dry weight of the embryonic and nodal root systems. Phenotypic variation explained by individual QTL ranged from 0.44 % (number of seminal roots, NyH population) to 13.5 % (shoot dry weight, OhW population). Identification of QTL for root architectural traits may be useful for developing genotypes that are better suited to specific soil environments.     

Quantitative trait loci controlling aluminium tolerance in two accessions of Arabidopsis thaliana (Landsberg erecta and Cape Verde Islands)

Quantitative trait loci (QTL) analysis of aluminium (Al) tolerance was performed using Ler/Cvi recombinant inbred (RI) lines of Arabidopsis thaliana. Relative root length (RRL) (root length with 4 µm Al/root length with no Al at pH 5.0) on day 5 was used as the Al tolerance index for QTL analysis. Al tolerance judged by RRL was well correlated to tolerance judged by other indexes, including accumulation of callose, reactive oxygen species in the root apex and growth performance on acid soil containing a large amount of exchangeable Al. Using data sets with an h_b² of 0.91, two QTLs were detected at the top of chromosome 1 and bottom of chromosome 3. These QTLs explained 40 and 16% of the phenotypic variation of Al tolerance, respectively, and the positive effect of the Cvi allele. The QTL on chromosome 1 overlapped with a major QTL in another recombinant inbred population, and is possibly related to malate excretion. A complete pair-wise search revealed 11 sets of epistatic interacting loci pairs, which accounted for the transgressive segregation among the RI population. Several epistatic interactions shared the same chromosomal region, indicating the possible involvement of regulatory proteins in Al tolerance in Arabidopsis.     

Quantitative trait loci analysis of primary cell wall composition in Arabidopsis

Quantitative trait loci (QTL) analysis was used to identify genes underlying natural variation in primary cell wall composition in Arabidopsis (Arabidopsis thaliana). The cell walls of dark-grown seedlings of a Bay-0 x Shahdara recombinant inbred line population were analyzed using three miniaturized global cell wall fingerprinting techniques: monosaccharide composition analysis by gas chromatography, xyloglucan oligosaccharide mass profiling, and whole-wall Fourier-transform infrared microspectroscopy. Heritable variation and transgression were observed for the arabinose-rhamnose ratio, xyloglucan side-chain composition (including O-acetylation levels), and absorbance for a subset of Fourier-transform infrared wavenumbers. In total, 33 QTL, corresponding to at least 11 different loci controlling dark-grown hypocotyl length, pectin composition, and levels of xyloglucan fucosylation and O-acetylation, were identified. One major QTL, accounting for 51% of the variation in the arabinose-rhamnose ratio, affected the number of arabinan side chains presumably attached to the pectic polysaccharide rhamnogalacturonan I, paving the way to positional cloning of the first gene underlying natural variation in pectin structure. Several QTL were found to be colocalized, which may have implications for the regulation of xyloglucan metabolism. These results demonstrate the feasibility of combining fingerprinting techniques, natural variation, and quantitative genetics to gain original insight into the molecular mechanisms underlying the structure and metabolism of cell wall polysaccharides.     

Quantitative trait loci for root-penetration ability and root thickness in rice: comparison of genetic backgrounds.

Drought is the major abiotic stress limiting rice (Oryza sativa) production and yield stability in rainfed lowland and upland ecosystems. Root systems play an important role in drought resistance. Incorporation of root selection criteria in drought resistance improvement is difficult due to lack of reliable and efficient screening techniques. Using a wax-petrolatum layer system simulated to compacted soil layers, root traits were evaluated in a doubled haploid (DH) population derived from the cross between 'IR64' and 'Azucena'. Twelve putative QTLs (quantitative trait loci) were detected by interval mapping comprising four QTLs for root-penetration ability, four QTLs for root thickness, two QTLs for penetrated root number, and two QTLs for total root number. These QTLs individually explained 8.4% to 16.4% of the phenotypic variation. No QTL was detected for maximum penetrated root length by interval mapping. One QTL located between RG104 and RG348 was found to influence both root-penetration ability and root thickness. QTLs for root-penetration ability and root thickness were compared across two populations, 'IR64'-'Azucena' and 'CO39'-'Moroberekan', and different testing conditions. The identified consistent QTLs could be used for marker-assisted selection for deep and thick roots with high root-penetration ability in rice.