Comparative mapping of quantitative trait loci sculpting the curd of Brassica oleracea

The enlarged inflorescence (curd) of cauliflower and broccoli provide not only a popular vegetable for human consumption, but also a unique opportunity for scientists who seek to understand the genetic basis of plant growth and development. By the comparison of quantitative trait loci (QTL) maps constructed from three different F(2) populations, we identified a total of 86 QTL that control eight curd-related traits in Brassica oleracea. The 86 QTL may reflect allelic variation in as few as 67 different genetic loci and 54 ancestral genes. Although the locations of QTL affecting a trait occasionally corresponded between different populations or between different homeologous Brassica chromosomes, our data supported other molecular and morphological data in suggesting that the Brassica genus is rapidly evolving. Comparative data enabled us to identify a number of candidate genes from Arabidopsis that warrant further investigation to determine if some of them might account for Brassica QTL. The Arabidopsis/Brassica system is an important example of both the challenges and opportunities associated with extrapolation of genomic information from facile models to large-genome taxa including major crops.     

Flowering time quantitative trait Loci analysis of oilseed brassica in multiple environments and genomewide alignment with Arabidopsis

Most agronomical traits exhibit quantitative variation, which is controlled by multiple genes and are environmentally dependent. To study the genetic variation of flowering time in Brassica napus, a DH population and its derived reconstructed F(2) population were planted in 11 field environments. The flowering time varied greatly with environments; 60% of the phenotypic variation was attributed to genetic effects. Five to 18 QTL at a statistically significant level (SL-QTL) were detected in each environment and, on average, two new SL-QTL were discovered with each added environment. Another type of QTL, micro-real QTL (MR-QTL), was detected repeatedly from at least 2 of the 11 environments; resulting in a total of 36 SL-QTL and 6 MR-QTL. Sixty-three interacting pairs of loci were found; 50% of them were involved in QTL. Hundreds of floral transition genes in Arabidopsis were aligned with the linkage map of B. napus by in silico mapping; 28% of them aligned with QTL regions and 9% were consistent with interacting loci. One locus, BnFLC10, in N10 and a QTL cluster in N16 were specific to spring- and winter-cropped environments respectively. The number of QTL, interacting loci, and aligned functional genes revealed a complex genetic network controlling flowering time in B. napus.     

Quantitative trait loci that control the oil content variation of rapeseed (Brassica napus L.)

Key message

This report describes an integrative analysis of seed-oil-content quantitative trait loci (QTL) in Brassica napus , using a high-density genetic map to align QTL among different populations.

Abstract

Rapeseed (Brassica napus) is an important source of edible oil and sustainable energy. Given the challenge involved in using only a few genes to substantially increase the oil content of rapeseed without affecting the fatty acid composition, exploitation of a greater number of genetic loci that regulate the oil content variation among rapeseed germplasm is of fundamental importance. In this study, we investigated variation in the seed-oil content among two related genetic populations of Brassica napus, the TN double-haploid population and its derivative reconstructed-F₂ population. Each population was grown in multiple experiments under different environmental conditions. Mapping of quantitative trait loci (QTL) identified 41 QTL in the TN populations. Furthermore, of the 20 pairs of epistatic interaction loci detected, approximately one-third were located within the QTL intervals. The use of common markers on different genetic maps and the TN genetic map as a reference enabled us to project QTL from an additional three genetic populations onto the TN genetic map. In summary, we used the TN genetic map of the B. napus genome to identify 46 distinct QTL regions that control seed-oil content on 16 of the 19 linkage groups of B. napus. Of these, 18 were each detected in multiple populations. The present results are of value for ongoing efforts to breed rapeseed with high oil content, and alignment of the QTL makes an important contribution to the development of an integrative system for genetic studies of rapeseed.     

Quantitative trait loci analysis of growth response to varying nitrogen sources in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Nitrogen absorption and assimilation is variable among plants as a result of two factors: the source of nitrogen available and the genetic variation among species within the resulting nitrogen pathways. Several genes involved in nitrogen cycling have been identified, yet little is known about the genes that control quantitative responses to different nitrogen sources. With quantitative trait loci (QTL) mapping in Arabidopsis thaliana recombinant inbred lines (Columbia x Landsberg erecta) we have identified chromosomal regions controlling aerial mass, root mass, and root length when plants are grown in nitrate, ammonium, ammonium nitrate, or low nitrogen treatments. A total of 16 QTL ( P < 0.01) were identified among the nitrogen treatments. Most of the QTL were specific to a single treatment. The percentage additive genetic effects of significant QTL were as high as 17%. Five significant QTL corresponded to the locations of candidate genes associated with nitrogen assimilation, while a few QTL corresponded with candidate genes in the developmental pathways. Most QTL were not shared across treatments, suggesting that there is no optimal genotype for all nitrogen sources.     

Characterization of metabolite quantitative trait loci and metabolic networks that control glucosinolate concentration in the seeds and leaves of Brassica napus

? Glucosinolates are a major class of secondary metabolites found in the Brassicaceae, whose degradation products are proving to be increasingly important for human health and in crop protection. ? The genetic and metabolic basis of glucosinolate accumulation was dissected through analysis of total glucosinolate concentration and its individual components in both leaves and seeds of a doubled-haploid (DH) mapping population of oilseed rape/canola (Brassica napus). ? The quantitative trait loci (QTL) that had an effect on glucosinolate concentration in either or both of the organs were integrated, resulting in 105 metabolite QTL (mQTL). Pairwise correlations between individual glucosinolates and prior knowledge of the metabolic pathways involved in the biosynthesis of different glucosinolates allowed us to predict the function of genes underlying the mQTL. Moreover, this information allowed us to construct an advanced metabolic network and associated epistatic interactions responsible for the glucosinolate composition in both leaves and seeds of B. napus. ? A number of previously unknown potential regulatory relationships involved in glucosinolate synthesis were identified and this study illustrates how genetic variation can affect a biochemical pathway.     

The identification and mapping of candidate genes and QTL involved in the fatty acid desaturation pathway in <Emphasis Type="Italic">Brassica napus</Emphasis>

We constructed a linkage map for the population QDH, which was derived from a cross between an oilseed rape cultivar and a resynthesised Brassica napus. The linkage map included ten markers linked to loci orthologous to those encoding fatty acid biosynthesis genes in Arabidopsis thaliana. The QDH population contains a high level of allelic variation, particularly in the C genome. We conducted quantitative trait locus (QTL) analyses, using field data obtained over 3 years, for the fatty acid composition of seed oil. The population segregates for the two major loci controlling erucic acid content, on linkage groups A8 and C3, which quantitatively affect the content of other fatty acids and is a problem generally encountered when crossing “wild” germplasm with cultivated “double low” oilseed rape cultivars. We assessed three methods for QTL analysis, interval mapping, multiple QTL mapping and single marker regression analysis of the subset of lines with low erucic acid. We found the third of these methods to be most appropriate for our main purpose, which was the study of the genetic control of the desaturation of 18-carbon fatty acids. This method enabled us to decouple the effect of the segregation of the erucic acid-controlling loci and identify 34 QTL for fatty acid content of seed oil, 14 in the A genome and 20 in the C genome. The QTL indicate the presence of 13 loci with novel alleles inherited from the progenitors of the resynthesised B. napus that might be useful for modulating the content or extent of desaturation of polyunsaturated fatty acids, only one of which coincides with the anticipated position of a candidate gene, an orthologue of FAD2.     

Novel loci control variation in reproductive timing in Arabidopsis thaliana in natural environments

Molecular biologists are rapidly characterizing the genetic basis of flowering in model species such as Arabidopsis thaliana. However, it is not clear how the developmental pathways identified in controlled environments contribute to variation in reproductive timing in natural ecological settings. Here we report the first study of quantitative trait loci (QTL) for date of bolting (the transition from vegetative to reproductive growth) in A. thaliana in natural seasonal field environments and compare the results with those obtained under typical growth-chamber conditions. Two QTL specific to long days in the chamber were expressed only in spring-germinating cohorts in the field, and two loci specific to short days in the chamber were expressed only in fall-germinating cohorts, suggesting differential involvement of the photoperiod pathway in different seasonal environments. However, several other photoperiod-specific QTL with large effects in controlled conditions were undetectable in natural environments, indicating that expression of allelic variation at these loci was overridden by environmental factors specific to the field. Moreover, a substantial number of QTL with major effects on bolting date in one or more field environments were undetectable under controlled environment conditions. These novel loci suggest the involvement of additional genes in the transition to flowering under ecologically relevant conditions.     

Selection on QTL and complex traits in complex environments

Understanding genetic variation for complex traits in heterogeneous environments is a fundamental problem in biology. In this issue of Molecular Ecology, Fournier‐Level et al. ( 2013 ) analyse quantitative trait loci (QTL) influencing ecologically important phenotypes in mapping populations of Arabidopsis thaliana grown in four habitats across its native European range. They used causal modelling to quantify the selective consequences of life history and morphological traits and QTL on components of fitness. They found phenology QTL colocalizing with known flowering time genes as well as novel loci. Most QTL influenced fitness via life history and size traits, rather than QTL having direct effects on fitness. Comparison of phenotypes among environments found no evidence for genetic trade‐offs for phenology or growth traits, but genetic trade‐offs for fitness resulted because flowering time had opposite fitness effects in different environments. These changes in QTL effects and selective consequences may maintain genetic variation among populations.     

Identification of Botrytis cinerea susceptibility loci in Arabidopsis thaliana

Botrytis cinerea is a major pathogen of fruit and vegetable crops causing both pre- and post-harvest grey mould. We have analysed 16 Arabidopsis thaliana ecotypes for natural variation in B. cinerea susceptibility. Susceptibility was associated with lower camalexin accumulation, and three ecotypes (Cape Verdi Islands (Cvi-0), Slavice (Sav-0) and Kindalville (Kin-0)) showed differential susceptibility to the two B. cinerea isolates used. Subsequently, to better understand the genetic control of grey mould disease, we assayed the Arabidopsis Landsberg erecta (Ler) x Columbia (Col-0) recombinant inbred population with the two isolates, and identified multiple small-to-medium-effect quantitative trait loci (QTL) governing susceptibility. Interestingly, the QTL for each isolate are distinct, suggesting that different mechanisms govern defence against these two isolates. Two QTL for each isolate exhibited epistatic interactions with specific allele combinations generating heightened B. cinerea susceptibility.     

Quantitative trait loci for flowering time and morphological traits in multiple populations of Brassica rapa

Wide variation for morphological traits exists in Brassica rapa and the genetic basis of this morphological variation is largely unknown. Here is a report on quantitative trait loci (QTL) analysis of flowering time, seed and pod traits, growth-related traits, leaf morphology, and turnip formation in B. rapa using multiple populations. The populations resulted from crosses between the following accessions: Rapid cycling, Chinese cabbage, Yellow sarson, Pak choi, and a Japanese vegetable turnip variety. A total of 27 QTL affecting 20 morphological traits were detected, including eight QTL for flowering time, six for seed traits, three for growth-related traits and 10 for leaf traits. One major QTL was found for turnip formation. Principal component analysis and co-localization of QTL indicated that some loci controlling leaf and seed-related traits and those for flowering time and turnip formation might be the same. The major flowering time QTL detected in all populations on linkage group R02 co-localized with BrFLC2. One major QTL, controlling turnip formation, was also mapped at this locus. The genes that may underly this QTL and comparative analyses between the four populations and with Arabidopsis thaliana are discussed.     

Major-effect alleles at relatively few loci underlie distinct vernalization and flowering variation in Arabidopsis accessions

We have explored the genetic basis of variation in vernalization requirement and response in Arabidopsis accessions, selected on the basis of their phenotypic distinctiveness. Phenotyping of F2 populations in different environments, plus fine mapping, indicated possible causative genes. Our data support the identification of FRI and FLC as candidates for the major-effect QTL underlying variation in vernalization response, and identify a weak FLC allele, caused by a Mutator-like transposon, contributing to flowering time variation in two N. American accessions. They also reveal a number of additional QTL that contribute to flowering time variation after saturating vernalization. One of these was the result of expression variation at the FT locus. Overall, our data suggest that distinct phenotypic variation in the vernalization and flowering response of Arabidopsis accessions is accounted for by variation that has arisen independently at relatively few major-effect loci.     

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.     

Activity and mRNA abundance of Delta-5 and Delta-6 fatty acid desaturases in two human cell lines

We analyzed fatty acid biosynthesis in Chang and ZR-75-1 cells. Both cell lines could desaturate and further elongate substrates for Delta-5 desaturase. ZR-75-1 but not Chang cells showed Delta-6 desaturation of 18:2n-6, 18:3n-3, 24:4n-6 and 24:5n-3. In both cell lines, the mRNA abundance can be related to Delta-5 or Delta-6 fatty acid desaturase activities. These results suggest that desaturase genes could have, at least in part, independent control mechanisms and that Delta-6 desaturase impairment is not specific to any particular step of the fatty acid metabolic pathways, which may diminish the rationale for the existence of at least two distinct enzymes.     

Mapping and cloning of FAD2 gene to develop allele-specific PCR for oleic acid in spring turnip rape (Brassica rapa ssp. oleifera)

The previously identified QTL for oleic acid content observed in an F2 population from the Brassica rapa ssp. oleifera cross Jo4002 × Jo4072 (a high-oleic-acid individual) was mapped more precisely by adding markers to the linkage group which harbours the locus. In addition, the fad2 gene, which is known to encode the 18:1 desaturase in Arabidopsis, was mapped in Brassica, too. The results are consistent with the QTL corresponding to the Arabidopsis fad2 gene. Comparison of the wild-type and high-oleic-acid allele of the locus revealed only one difference in their nucleic acid sequences leading to an amino acid change. This substitution of leucine by proline most likely affects the fold of the protein and thereby activity of the enzyme. Using this base difference, an allele-specific PCR was designed. The allele-specific markers will be very effective in selection for plants with high-oleic-acid content derived from Jo4072 because they are located exactly at the locus and can differentiate between homo- and heterozygotes.     

Identification of a gene that complements an Arabidopsis mutant deficient in chloroplast omega 6 desaturase activity.

Membrane lipids of the fad6 (formerly fadC) mutant of Arabidopsis, which is deficient in chloroplast omega 6 desaturase activity, have increased levels of monounsaturated fatty acids and are deficient in trienoic fatty acids. A putative fad6 cDNA clone was isolated by probing a cDNA library with a degenerate oligonucleotide based on a conserved region within known omega 3 desaturase genes. Expression of the cDNA in transgenic plants of a fad6 mutant restored normal levels of all fatty acids. When used as a hybridization probe, the cDNA identified a restriction fragment-length polymorphism that co-segregated with the fad6 mutation. Thus, on the basis of a genetic complementation test and genetic map position, the fad6 gene is encoded by the cDNA. The cDNA encoded a 418-amino acid polypeptide of 47,727 D that displayed a high degree of sequence similarity to a delta 12 desaturase from the cyanobacterium Synechocystis. The fad6 gene exhibited less sequence homology to any known higher plant desaturase, including an endoplasmic reticulum-localized omega 6 desaturase corresponding to the Arabidopsis fad2 gene.     

Quantitative trait loci for locomotor behavior in Drosophila melanogaster

Locomotion is an integral component of most animal behaviors and many human diseases and disorders are associated with locomotor deficits, but little is known about the genetic basis of natural variation in locomotor behavior. Locomotion is a complex trait, with variation attributable to the joint segregation of multiple interacting quantitative trait loci (QTL), with effects that are sensitive to the environment. We assessed variation in a component of locomotor behavior (locomotor reactivity) in a population of 98 recombinant inbred lines of Drosophila melanogaster and mapped four QTL affecting locomotor reactivity by linkage to polymorphic roo transposable element insertion sites. We used complementation tests of deficiencies to fine map these QTL to 12 chromosomal regions and complementation tests of mutations to identify 13 positional candidate genes affecting locomotor reactivity, including Dopa decarboxylase (Ddc), which catalyzes the final step in the synthesis of serotonin and dopamine. Linkage disequilibrium mapping in a population of 164 second chromosome substitution lines derived from a single natural population showed that polymorphisms at Ddc were associated with naturally occurring genetic variation in locomotor behavior. These data implicate variation in the synthesis of bioamines as a factor contributing to natural variation in locomotor reactivity.     

Expression of the Isochrysis C18-delta9 polyunsaturated fatty acid specific elongase component alters Arabidopsis glycerolipid profiles

A cDNA isolated from the prymnesiophyte micro-alga Isochrysis galbana, designated IgASE1, encodes a fatty acid elongating component that is specific for linoleic acid (C18:2n-6) and alpha-linolenic acid (C18:3n-3). Constitutive expression of IgASE1 in Arabidopsis resulted in the accumulation of eicosadienoic acid (EDA; C20:2n-6) and eicosatrienoic acid (ETrA; C20:3n-3) in all tissues examined, with no visible effects on plant morphology. Positional analysis of the various lipid classes indicated that these novel fatty acids were largely excluded from the sn-2 position of chloroplast galactolipids and seed triacylglycerol, whereas they were enriched in the same position in phosphatidylcholine. EDA and ETrA are precursors of arachidonic acid (C20:4n-6), eicosapentaenoic acid (C20:5n-3), and docosahexaenoic acid (C22:6n-3) synthesized via the so-called omega6 Delta8 desaturase and omega3 Delta8 desaturase biosynthetic pathways, respectively. The synthesis of significant quantities of EDA and ETrA in a higher plant is therefore a key step in the production of very long chain polyunsaturated fatty acid in oil-seed species. The results are further discussed in terms of prokaryotic and eukaryotic pathways of lipid synthesis in plants.     

Identification of quantitative trait loci that regulate Arabidopsis root system size and plasticity

Root system size (RSS) is a complex trait that is greatly influenced by environmental cues. Hence, both intrinsic developmental pathways and environmental-response pathways contribute to RSS. To assess the natural variation in both types of pathways, we examined the root systems of the closely related Arabidopsis ecotypes Landsberg erecta (Ler) and Columbia (Col) grown under mild osmotic stress conditions. We found that Ler initiates more lateral root primordia, produces lateral roots from a higher percentage of these primordia, and has an overall larger root system than Col under these conditions. Furthermore, although each of these parameters is reduced by osmotic stress in both ecotypes, Ler shows a decreased sensitivity to osmotica. To understand the genetic basis for these differences, QTL for RSS under mild osmotic stress were mapped in a Ler x Col recombinant inbred population. Two robust quantitative trait loci (QTL) were identified and confirmed in near-isogenic lines (NILs). The NILs also allowed us to define distinct physiological roles for the gene(s) at each locus. This study provides insight into the genetic and physiological complexity that determines RSS and begins to dissect the molecular basis for naturally occurring differences in morphology and developmental plasticity in the root system.     

Genetic and physical mapping of flowering time loci in canola (Brassica napus L.)

We identified quantitative trait loci (QTL) underlying variation for flowering time in a doubled haploid (DH) population of vernalisation—responsive canola (Brassica napus L.) cultivars Skipton and Ag-Spectrum and aligned them with physical map positions of predicted flowering genes from the Brassica rapa genome. Significant genetic variation in flowering time and response to vernalisation were observed among the DH lines from Skipton/Ag-Spectrum. A molecular linkage map was generated comprising 674 simple sequence repeat, sequence-related amplified polymorphism, sequence characterised amplified region, Diversity Array Technology, and candidate gene based markers loci. QTL analysis indicated that flowering time is a complex trait and is controlled by at least 20 loci, localised on ten different chromosomes. These loci each accounted for between 2.4 and 28.6 % of the total genotypic variation for first flowering and response to vernalisation. However, identification of consistent QTL was found to be dependant upon growing environments. We compared the locations of QTL with the physical positions of predicted flowering time genes located on the sequenced genome of B. rapa. Some QTL associated with flowering time on A02, A03, A07, and C06 may represent homologues of known flowering time genes in Arabidopsis; VERNALISATION INSENSITIVE 3, APETALA1, CAULIFLOWER, FLOWERING LOCUS C, FLOWERING LOCUS T, CURLY LEAF, SHORT VEGETATIVE PHASE, GA3 OXIDASE, and LEAFY. Identification of the chromosomal location and effect of the genes influencing flowering time may hasten the development of canola varieties having an optimal time for flowering in target environments such as for low rainfall areas, via marker-assisted selection.