Development of an efficient glucosinolate extraction method.

BACKGROUND: Glucosinolates, anionic sulfur rich secondary metabolites, have been extensively studied because of their occurrence in the agriculturally important brassicaceae and their impact on human and animal health. There is also increasing interest in the biofumigant properties of toxic glucosinolate hydrolysis products as a method to control agricultural pests. Evaluating biofumigation potential requires rapid and accurate quantification of glucosinolates, but current commonly used methods of extraction prior to analysis involve a number of time consuming and hazardous steps; this study aimed to develop an improved method for glucosinolate extraction. RESULTS: Three methods previously used to extract glucosinolates from brassicaceae tissues, namely extraction in cold methanol, extraction in boiling methanol, and extraction in boiling water were compared across tissue type (root, stem leaf) and four brassicaceae species (B. juncea, S. alba, R. sativus, and E. sativa). Cold methanol extraction was shown to perform as well or better than all other tested methods for extraction of glucosinolates with the exception of glucoraphasatin in R. sativus shoots. It was also demonstrated that lyophilisation methods, routinely used during extraction to allow tissue disruption, can reduce final glucosinolate concentrations and that extracting from frozen wet tissue samples in cold 80% methanol is more effective. CONCLUSIONS: We present a simplified method for extracting glucosinolates from plant tissues which does not require the use of a freeze drier or boiling methanol, and is therefore less hazardous, and more time and cost effective. The presented method has been shown to have comparable or improved glucosinolate extraction efficiency relative to the commonly used ISO method for major glucosinolates in the Brassicaceae species studied: sinigrin and gluconasturtiin in B. juncea; sinalbin, glucotropaeolin, and gluconasturtiin in S. alba; glucoraphenin and glucoraphasatin in R. sativus; and glucosatavin, glucoerucin and glucoraphanin in E. sativa.

Root morphology and seed and leaf ionomic traits in a Brassica napus L. diversity panel show wide phenotypic variation and are characteristic of crop habit.

BACKGROUND: Mineral nutrient uptake and utilisation by plants are controlled by many traits relating to root morphology, ion transport, sequestration and translocation. The aims of this study were to determine the phenotypic diversity in root morphology and leaf and seed mineral composition of a polyploid crop species, Brassica napus L., and how these traits relate to crop habit. Traits were quantified in a diversity panel of up to 387 genotypes: 163 winter, 127 spring, and seven semiwinter oilseed rape (OSR) habits, 35 swede, 15 winter fodder, and 40 exotic/unspecified habits. Root traits of 14 d old seedlings were measured in a 'pouch and wick' system (n = ~24 replicates per genotype). The mineral composition of 3-6 rosette-stage leaves, and mature seeds, was determined on compost-grown plants from a designed experiment (n = 5) by inductively coupled plasma-mass spectrometry (ICP-MS). RESULTS: Seed size explained a large proportion of the variation in root length. Winter OSR and fodder habits had longer primary and lateral roots than spring OSR habits, with generally lower mineral concentrations. A comparison of the ratios of elements in leaf and seed parts revealed differences in translocation processes between crop habits, including those likely to be associated with crop-selection for OSR seeds with lower sulphur-containing glucosinolates. Combining root, leaf and seed traits in a discriminant analysis provided the most accurate characterisation of crop habit, illustrating the interdependence of plant tissues. CONCLUSIONS: High-throughput morphological and composition phenotyping reveals complex interrelationships between mineral acquisition and accumulation linked to genetic control within and between crop types (habits) in B. napus. Despite its recent genetic ancestry (<10 ky), root morphology, and leaf and seed composition traits could potentially be used in crop improvement, if suitable markers can be identified and if these correspond with suitable agronomy and quality traits.

Towards the genetic architecture of seed lipid biosynthesis and accumulation in Arabidopsis thaliana.

We report the quantitative genetic analysis of seed oil quality and quantity in six Arabidopsis thaliana recombinant inbred populations, in which the parent accessions were from diverse geographical origins, and were selected on the basis of variation for seed oil content and lipid composition. Although most of the biochemical steps involved in lipid biosynthesis are known and the key genes have been identified, the regulation of the processes that results in the final oil composition and total amount is not understood. By using physically anchored markers it was possible to compare results across populations. A total of 219 quantitative trait loci (QTLs) were identified, of which 81 were significant at P<0.001. Some of these colocalise with QTLs identified previously, but many novel QTLs were also identified. The results highlight the importance of studying traits in multiple populations, which will lead to a better understanding of the contribution that natural variation makes to the genetic architecture of a phenotype.

The identification and mapping of candidate genes and QTL involved in the fatty acid desaturation pathway in Brassica napus.

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.

A comparative linkage map of oilseed rape and its use for QTL analysis of seed oil and erucic acid content.

We have developed a new DH mapping population for oilseed rape, named TNDH, using genetically and phenotypically diverse parental lines. We used the population in the construction of a high stringency genetic linkage map, consisting of 277 loci, for use in quantitative genetic analysis. A proportion of the markers had been used previously in the construction of linkage maps for Brassica species, thus permitting the alignment of maps. The map includes 68 newly developed Sequence Tagged Site (STS) markers targeted to the homologues of defined genes of A. thaliana. The use of these markers permits the alignment of our linkage map with the A. thaliana genome sequence. An additional 74 loci (31 newly developed STS markers and 43 loci defined by SSR and RFLP markers that had previously been used in published linkage maps) were added to the map. These markers increased the resolution of alignment of the newly constructed linkage map with existing Brassica linkage maps and the A. thaliana genome sequence. We conducted field trials with the TNDH population at two sites, and over 2 years, and identified reproducible QTL for seed oil content and erucic acid content. The results provide new insights into the genetic control of seed oil and erucic acid content in oilseed rape, and demonstrate the utility of the linkage map and population.

Physical mapping and microsynteny of Brassica rapa ssp. pekinensis genome corresponding to a 222 kbp gene-rich region of Arabidopsis chromosome 4 and partially duplicated on chromosome 5.

We constructed a bacterial artificial chromosome (BAC) library, designated as KBrH, from high molecular weight genomic DNA of Brassica rapa ssp. pekinensis (Chinese cabbage). This library, which was constructed using HindIII-cleaved genomic DNA, consists of 56,592 clones with average insert size of 115 kbp. Using a partially duplicated DNA sequence of Arabidopsis, represented by 19 and 9 predicted genes on chromosome 4 and 5, respectively, and BAC clones from the KBrH library, we studied conservation and microsynteny corresponding to the Arabidopsis regions in B. rapa ssp. pekinensis. The BAC contigs assembled according to the Arabidopsis homoeologues revealed triplication and rearrangements in the Chinese cabbage. In general, collinearity of genes in the paralogous segments was maintained, but gene contents were highly variable with interstitial losses. We also used representative BAC clones, from the assembled contigs, as probes and hybridized them on mitotic (metaphase) and/or meiotic (leptotene/pachytene/metaphase I) chromosomes of Chinese cabbage using bicolor fluorescence in situ hybridization. The hybridization pattern physically identified the paralogous segments of the Arabidopsis homoeologues on B. rapa ssp. pekinensis chromosomes. The homoeologous segments corresponding to chromosome 4 of Arabidopsis were located on chromosomes 2, 8 and 7, whereas those of chromosome 5 were present on chromosomes 6, 1 and 4 of B. rapa ssp. pekinensis.

Conservation of microstructure between a sequenced region of the genome of rice and multiple segments of the genome of Arabidopsis thaliana.

The nucleotide sequence was determined for a 340-kb segment of rice chromosome 2, revealing 56 putative protein-coding genes. This represents a density of one gene per 6.1 kb, which is higher than was reported for a previously sequenced segment of the rice genome. Sixteen of the putative genes were supported by matches to ESTs. The predicted products of 29 of the putative genes showed similarity to known proteins, and a further 17 genes showed similarity only to predicted or hypothetical proteins identified in genome sequence data. The region contains a few transposable elements: one retrotransposon, and one transposon. The segment of the rice genome studied had previously been identified as representing a part of rice chromosome 2 that may be homologous to a segment of Arabidopsis chromosome 4. We confirmed the conservation of gene content and order between the two genome segments. In addition, we identified a further four segments of the Arabidopsis genome that contain conserved gene content and order. In total, 22 of the 56 genes identified in the rice genome segment were represented in this set of Arabidopsis genome segments, with at least five genes present, in conserved order, in each segment. These data are consistent with the hypothesis that the Arabidopsis genome has undergone multiple duplication events. Our results demonstrate that conservation of the genome microstructure can be identified even between monocot and dicot species. However, the frequent occurrence of duplication, and subsequent microstructure divergence, within plant genomes may necessitate the integration of subsets of genes present in multiple redundant segments to deduce evolutionary relationships and identify orthologous genes.

Duplicate and diverge: the evolution of plant genome microstructure.

The use of positional approaches for the isolation of genes from most crop species is difficult due to the large size of their genomes. If the order of genes in segments of the genomes is similar in different plants, it might be feasible to use smaller genomes as templates upon which to base strategies for the positional cloning of genes from other species. Comparative genetic mapping, using markers such as restriction-fragment length polymorphisms, has revealed extensive conservation of long-range genome organization (macrostructure) between related species. But is the organization of the tens or hundreds of genes between the genetic markers also conserved? Recent results suggest that the fine-scale structure (microstructure) of plant genomes is more dynamic than previously assumed from investigations of the macrostructure.

Comparative physical mapping of segments of the genome of Brassica oleracea var. alboglabra that are homoeologous to sequenced regions of chromosomes 4 and 5 of Arabidopsis thaliana.

Due to their relatedness to Arabidopsis thaliana (Arabidopsis), the cultivated Brassica species represent the first group of crops with which to evaluate comparative genomics approaches to understanding biological processes and manipulating traits. We have constructed a high-quality binary BAC library (JBo) from genomic DNA of Brassica oleracea var. alboglabra, in order to underpin such investigations. Using the Arabidopsis genome sequence and clones from the JBo library, we have analysed aspects of gene conservation and microsynteny between a 222 kb region of the genome of Arabidopsis and homoeologous segments of the genome of B. oleracea. All 19 predicted genes tested were found to hybridize to clones in the JBo library, indicating a high level of gene conservation. Further analyses and physical mapping with the BAC clones identified allowed us to construct clone contig maps and analyse in detail the gene content and organization in the set of paralogous segments identified in the genome of B. oleracea. Extensive divergence of gene content was observed, both between the B. oleracea paralogous segments and between them and their homoeologous segment within the genome of Arabidopsis. However, the genes present show highly conserved collinearity with their orthologues in the genome of Arabidopsis. We have identified one example of a Brassica gene in a non-collinear position and one rearrangement. Some of the genes not present in the discernible homoeologous regions appear to be located elsewhere in the B. oleracea genome. The implications of our findings for comparative map-based cloning of genes from crop species are discussed.

Insights into the structural and functional evolution of plant genomes afforded by the nucleotide sequences of chromosomes 2 and 4 of Arabidopsis thaliana.

The rapidly accumulating genome sequence data from the plant Arabidopsis thaliana allows more detailed analysis of genome content and organisation than ever before possible in plants. The genome shows a surprisingly high level of genetic redundancy, with as many as 75% of gene products showing significant homology to another protein of A. thaliana. Many duplicated genes occur in arrays of conserved order and indicate that A. thaliana is likely to have had a tetraploid ancestor. Analysis of the divergence of duplicated genome segments leads to the prediction of two major modes of plant genome evolution: macro-scale duplication and rearrangement of chromosomes and micro-scale translocations, duplication and loss of individual genes or small groups of genes.

Progress in Arabidopsis genome sequencing and functional genomics.

Arabidopsis thaliana has a relatively small genome of approximately 130 Mb containing about 10% repetitive DNA. Genome sequencing studies reveal a gene-rich genome, predicted to contain approximately 25000 genes spaced on average every 4.5 kb. Between 10 to 20% of the predicted genes occur as clusters of related genes, indicating that local sequence duplication and subsequent divergence generates a significant proportion of gene families. In addition to gene families, repetitive sequences comprise individual and small clusters of two to three retroelements and other classes of smaller repeats. The clustering of highly repetitive elements is a striking feature of the A. thaliana genome emerging from sequence and other analyses.

Identification and analysis of homoeologous segments of the genomes of rice and Arabidopsis thaliana.

Using contiguous genomic DNA sequences of Arabidopsis thaliana, we were able to identify a region of conserved structure in the genome of rice. The conserved, and presumptive homoeologous segments, are 194 kb and 219-300 kb in size in Arabidopsis and rice, respectively. They contain five homologous genes, distinguished in order by a single inversion. These represent the first homoeologous segments identified in the genomes of a dicot and a monocot, demonstrating that fine-scale conservation of genome structure exists and is detectable across this major divide in the angiosperms. The conserved framework of genes identified is interspersed with non-conserved genes, indicating that mechanisms beyond segmental inversions and translocations need to be invoked to fully explain plant genome evolution, and that the benefits of comparative genomics over such large taxonomic distances may be limited.

Clearing a path through the jungle: progress in Arabidopsis genomics.

Progress in sequencing the genome of the model plant Arabidopsis is reviewed. The resulting analysis of the sequence indicates an information-rich genome that is being tackled by a variety of high-throughput approaches aimed at understanding the functions of plant genes. The information derived from these systematic studies is providing important new knowledge of biological processes found uniquely in plants for comparison with that obtained in other multicellular organisms.

Sequence and analysis of chromosome 4 of the plant Arabidopsis thaliana.

The higher plant Arabidopsis thaliana (Arabidopsis) is an important model for identifying plant genes and determining their function. To assist biological investigations and to define chromosome structure, a coordinated effort to sequence the Arabidopsis genome was initiated in late 1996. Here we report one of the first milestones of this project, the sequence of chromosome 4. Analysis of 17.38 megabases of unique sequence, representing about 17% of the genome, reveals 3,744 protein coding genes, 81 transfer RNAs and numerous repeat elements. Heterochromatic regions surrounding the putative centromere, which has not yet been completely sequenced, are characterized by an increased frequency of a variety of repeats, new repeats, reduced recombination, lowered gene density and lowered gene expression. Roughly 60% of the predicted protein-coding genes have been functionally characterized on the basis of their homology to known genes. Many genes encode predicted proteins that are homologous to human and Caenorhabditis elegans proteins.

BAC representation of two low-copy regions of the genome of Arabidopsis thaliana.

Two regions of Arabidopsis chromosome 4, totalling 4.7 Mb, were assayed for representation in the TAMU and IGF BAC libraries. A directed approach to BAC identification was developed. Gel-purified DNA samples of YACs selected from the YAC-based physical map of chromosome 4 were used to probe high-density colony arrays of the BAC libraries. Strategies were developed that allowed the efficient construction of restriction maps and BAC contigs. Four hundred and sixty-four BACs were mapped, assembled into two complete contigs and used to analyse genomic representation. These BACs provided a mean of 9.4-fold redundant coverage, with a range of 2- to 22-fold. The representation provided by the two libraries showed almost coincident peaks and troughs, with a periodicity of approximately 200 kb. These results demonstrate that, provided both TAMU and IGF libraries are used in their entirety, BACs should provide an excellent resource for both physical mapping and sequencing of the Arabidopsis genome.

Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana.

The plant Arabidopsis thaliana (Arabidopsis) has become an important model species for the study of many aspects of plant biology. The relatively small size of the nuclear genome and the availability of extensive physical maps of the five chromosomes provide a feasible basis for initiating sequencing of the five chromosomes. The YAC (yeast artificial chromosome)-based physical map of chromosome 4 was used to construct a sequence-ready map of cosmid and BAC (bacterial artificial chromosome) clones covering a 1.9-megabase (Mb) contiguous region, and the sequence of this region is reported here. Analysis of the sequence revealed an average gene density of one gene every 4.8 kilobases (kb), and 54% of the predicted genes had significant similarity to known genes. Other interesting features were found, such as the sequence of a disease-resistance gene locus, the distribution of retroelements, the frequent occurrence of clustered gene families, and the sequence of several classes of genes not previously encountered in plants.

FCA, a gene controlling flowering time in Arabidopsis, encodes a protein containing RNA-binding domains.

A strong promoter of the transition to flowering in Arabidopsis is encoded by FCA. FCA has been cloned and shown to encode a protein containing two RNA-binding domains and a WW protein interaction domain. This suggests that FCA functions in the posttranscriptional regulation of transcripts involved in the flowering process. The FCA transcript is alternatively spliced with only one form encoding the entire FCA protein. Plants carrying the FCA gene fused to the strong constitutive 35S promoter flowered earlier, and the ratio and abundance of the different FCA transcripts were altered. Thus, FCA appears to be a component of a posttranscriptional cascade involved in the control of flowering time.

Transposition pattern of the maize element Ds in Arabidopsis thaliana.

As part of establishing an efficient transposon tagging system in Arabidopsis using the maize elements Ac and Ds, we have analyzed the inheritance and pattern of Ds transposition in four independent Arabidopsis transformants. A low proportion (33%) of plants inheriting the marker used to monitor excision contained a transposed Ds. Selection for the transposed Ds increased this to at least 49%. Overall, 68% of Ds transpositions inherited with the excision marker were to genetically linked sites; however, the distribution of transposed elements varied around the different donor sites. Mapping of transposed Ds elements that were genetically unlinked to the donor site showed that a proportion (3 of 11 tested) integrated into sites which were still physically linked.

Factors affecting the excision frequency of the maize transposable element Ds in Arabidopsis thaliana.

A two-element transposon system based on the maize elements Ac and Ds is currently being used for insertional mutagenesis in Arabidopsis. With the aim of making this system as efficient as possible we have continued to analyse several parameters which affect Ds activity in Arabidopsis. The influence of genomic position on Ds excision has been analysed in five lines carrying Ds integrated in different genomic locations. Differences in both somatic and germinal excision were observed between the different lines. The relationship between somatic and germinal excision, the timing of excision events and environmental influences on transposition frequency have been investigated. The effect of varying dosage of the different elements was also analysed. A strong positive dosage effect was observed for the transposase source, but not for the Ds element. Analysis of germinal excision events showed that the majority of them occurred very late in the development of the plant, resulting in the majority of Ds transpositions being independent events.

Heterologous transposon tagging of the DRL1 locus in Arabidopsis.

The development of heterologous transposon tagging systems has been an important objective for many laboratories. Here, we demonstrate the use of a Dissociation (Ds) derivative of the maize transposable element Activator (Ac) to tag the DRL1 locus of Arabidopsis. The drl1 mutant shows highly abnormal development with stunted roots, few root hairs, lanceolate leaves, and a highly enlarged, disorganized shoot apex that does not produce an inflorescence. The mutation was shown to be tightly linked to a transposed Ds, and somatic instability was observed in the presence of the transposase source. Some plants showing somatic reversion flowered and produced large numbers of wild-type progeny. These revertant progeny always inherited a DRL1 allele from which Ds had excised. Analysis of the changes in DNA sequence induced by the insertion and excision of the Ds element showed that they were typical of those induced by Ac and Ds in maize.