Natural variation in arabidopsis thaliana : resources for functional analysis (NATURAL)

Recombinant Inbred lines (F8 and higher generations) were obtained from the crosses between the following Arabidopsis accessions: Ler x Sha (Shakhdara) - 114 lines Ler x An-1 (Antwerp 1) -120 lines Ler x Kas-2 (Kashmir) - 164 lines Ler x Kond (Kondara) - 120 lines In addition a set of 80 introgression of different length covering the complete Cvi genome into Ler was obtained.

Complex traits like human disease, growth rate or crop yield are highly polygenic. Typically, few major and many minor QTL contribute to such traits. While progress has been made in identifying large-effect QTL, experimental constraints have limited knowledge of small-effect QTL. Therefore, a 1 centiMorgan segment of the Arabidopsis genome was chosen without knowledge about its effect on plant growth rate. This segment was dissected in a series of crosses between near-isogenic Arabidopsis lines that harbored recombination breakpoints in this genome segment, and the progeny of these crosses was investigated for variation in biomass accumulation. Plant lines were selected such that progeny from each cross segregated for a very small fragment of this genome segment, while flanking genomic regions were identical. Within 1 centiMorgan region, two small-effect growth rate QTL were detected, and one was identified as a serine-threonine protein kinase. Both QTL interacted epistatically with their genetic background. Futhermore, a signature of balancing selection was detected for the serine-threonine protein kinase gene. These results, if typical for the entire genome, suggest a highly polygenic, epistatic architecture of complex traits, with cryptic genetic variation maintained in part by balancing selection.

Seven QTLs affecting freezing tolerance have been identified by QTL mapping in the Ler/Cvi RIL population.Two QTLs (FTQ4 and FTQ6) showed effect under long and short day photoperiod, while the remainign five loci appear as photoperiod-dependent. Molecular analyses suggest that the CBF2 gene encoding an AP2-like transcription factor underlies the largest effect QTL, FTQ4. A 1.6 Kb deletion in the Cvi promoter region of CBF2 is suggested as the functional polymorphism causing a strong reduction of RNA expression.

Fourteen quantitative trait loci (QTLs) contributing to the large amount of natural variation for flowering time of Arabidopsis have been identified and mapped by analysing 5 populations of introgression lines and 2 new populations of recombinant inbred lines. One locus named as Flowering Arabidopsis QTL 1 (FAQ1) has been cloned and found to encode a putative trasncription factor. Genetic variation in FAQ1 orthologous genes in other speceis might be of interest to manipulate flowering time in crops. Two additional loci called FAQ2 and FAQ3 have been fine mapped. FAQ2 has been found to be a complex QTL consisting of at least two closely linked loci.

Two population of F8 recombinant inbred lines have been obtained from the crosses of Ler x Fei (230 RILs) and Ler x Ll-0 (135). These have been genotyped and their genetic maps obtained. Five populations of introgression lines carrying either early or late flwoering alleles from the accesssions Fuk, Ri-0, Kas-2, Shak or C24 in a Ler genetic background (a total of 100 introgression lines).

Growth rate of plant s was analyzed in two RIL populations, and genetic loci affecting growth have been identified. Near isogenic lines containing the loci of interest were made for future analyses of these genes controlling growth rate. Carbohydrate contents of leaves were analysed in order to reveal correlations between this parameter and growth rate. Growth appeared to be correlated with the accumulation of transient starch in the leaves. The rate of photosynthesis in a wide range of accessions (ecotypes) appeared to be very constant, with one noticeable exception. This deviating behaviour turned out to be due to herbicide resistance of this accession, which is likely to be man-induced. We exploited natural variation in Arabidopsis to unravel the genetics of accumulation of micronutrients (minerals) and anti-nutrional factors, viz. phytate. Loci for accumulation of phytate and phosphate, iron, zinc, and other minerals were found. The loci controlling phytate accumulation appeared to be different from those affecting iron and zinc, indicating that breeding for increased mineral content is feasible without interfering accumulation of phytate. Under stress (cold or salt) conditions, growth was severely hampered. The hypothesis that this reduction in growth was due to altered carbohydrate metabolism could not be substantiated.

The Arabidopsis thaliana cross C24 x Col, the F1 hybrids and c. 400 derived recombinant inbred lines (RILs) have been analysed for biomass accumulation, metabolite and element composition, trancript profiles, and 13C and 15N discrimination. Through QTL and gene expression analyses, genome segments and candidate genes have been identified (potentially) affecting these traits.

The Nottingham group has focused on assessing genetic variation in Arabidopsis for nutritionally important vitamins, ascorbate (vitamin C) and folate. Little variation was seen amongst Arabidopsis accessions in leaf ascorbate levels, but transgression was observed in the Ler/Cvi RI population, enabling 3 QTL to be identified, one of which co-localises with the VTC2 gene that exhibits expression differences between Cvi and Ler parents; and whose mutant allele exhibits dosage effects on ascorbate levels (Garratt et al, 2005a). Folate studies have focused on measuring variation in the degree of vitamin polyglutamation, a key determinant in folate bioavailability in the human diet, necessitating the development of an innovative LC-MS/MS assay (Garratt et al, 2005b). Our studies have revealed that several distinctly sized glutamated classes of folate exist in plant tissues which will negatively impact bio availability. Due to the low throughput nature of the LC-MS/MS method, a higher throughput screen at the seedling stage was developed, employing the anti-folate methatrexate (MTX). At least 5 QTL in the Ler/Cvi NIL population have been identified to confer enhanced resistance to the anti-folate methatrexate. Several folate related candidate genes are known to map in these introgressed regions. The impact of these QTL on folate composition is being characterised employing the LC-MS/MS method to reveal the basis of the MTX resistance phenotype, and ultimately pinpoint genes suitable for manipulating folate abundance and/or composition in crops.

Glucosinolates are ecologically important plant secondary compounds from the Brassicales/Capparales. They contribute to the quality and health characteristics of crop plants. The genetics of glucosinolate biosynthesis and variation was investigated in several Arabidopsis ecotypes and in progeny from crosses between Arabidopsis ecotypes. One major QTL was precisely identified which controls glucosinolate profiles and resistance to generalist insect herbivores. A single enzyme-encoding locus determines the side chain length of methionine-derived (aliphatic) glucosinolates in Arabidopsis, and contributes to resistance against generalist insect herbivores. Statistical methods of molecular population genetics detected a signiture of balancing selection for one of the genes encoded at this locus, maintaining unusually high levels of nucleotide and amino acid polymorphism at intermediate frequency in Arabidopsis thaliana. These results may help manipulate glucosinolate profiles in cruciferous crop plants, either by transgenic approaches or by marker-assisted breeding.

Ten recombinant inbred line (RIL) populations were generated for the Arabidopsis thaliana accession crosses Col x C24 (233 lines, F9), C24 x Col (249 lines, F9), Nd x C24 (248 lines, F8), C24 x Nd (250 lines, F8), Bch-1 x C24 (248 lines, F8), C24 x Bch-1 (249 lines, F8), Ak-1 x C24 (232 lines, F8), C24 x Ak-1 (224 lines, F8), Ler x C24 (250 lines, F5), C24 x Ler (249 lines, F5). The Col x C24 and C24 x Col lines have been genotyped with a framework marker set of 112 SNP markers, which has been made available for general use. Furthermore, two sets of genetic substitution lines (nearly isogenic lines, NILs) were created and genotyped: 80 lines of Col (recurrent parent) / C24 (donor parent) and 64 lines of C24 (recurrent parent) / Col (donor parent).

The genetics of seed dormancy was studied in 7 recombinant inbred line populations and revealed map positions for 12 QTls. For one locus (DOG1) the underlying gen was cloned by a combination of fine mapping and mutant approaches. This gene encodes a bZIP transcription factor and knock out mutants show except for a complete lack of dormancy no other plant phenotype. This specificity makes it possible to use such a gen for the modification of seed dormancy either by marker assisted selection or by transgenic approaches.

Biomass accumulation and nitrogen (N) metabolism related traits were measured on a population of 415 recombinant inbred lines derived from a cross between the Bay-0 and Shahdara ecotypes. Plants were grown in controlled culture chambers with limiting or non-limiting N supply. Many genetic loci were identified, showing genotype X environment interactions and co-localisations of QTL have revealed interesting links between different parameters. Heterogenous inbred family approach was used to validate four QTL and fine mapping experiments have been undertaken to clone the gene that is responsible for the variation at one of these QTL. In addition, anions and carbohydrates contents were analysed in leaves of plants grown under these two N conditions. Loci controlling soluble sugars and starch accumulation were identified and shown in some cases to be co-localised with QTL for N-related traits. This study allowed us to analyse the interactions between C and N metabolisms through a novel quantitative genetic approach.

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