Low Glucosinolate Lines Research Articles

Targeted silencing of genes in polyploids: lessons learned from Brassica juncea-glucosinolate system.

Intron-spliced hairpin RNAi construct targeting the exonic region of BjuMYB28 driven by the native promoter is the best suited strategy for developing viable low glucosinolate lines in polyploid Brassica juncea. Targeted silencing of specific homolog(s) of a multigene family in polyploids through RNA interference (RNAi) is a challenging effort. Indian oilseed mustard (Brassica juncea), a natural allotetraploid, is expected to have 4-6 copies of every Arabidopsis gene ortholog. In the current study, we have attempted to establish the best gene silencing system suitable for BjuMYB28, a transcription factor gene, with the objective of developing low seed glucosinolate linesin B. juncea. After comparing multiple combinations of BjuMYB28 gene homologs, promoters, target regions (exon and 3' UTR) and silencing strategies (RNAi and antisense), we suggest that the intron-spliced hairpin RNAi construct targeting the specific exonic region of the BjuMYB28 gene under the control of native promoter, whose peak activity synchronises with the highest glucosinolate accumulation phase of the plant, is the best suited strategy for developing viable low glucosinolate lines in polyploid B. juncea.

Introgression of allelic diversity from genetically distinct variants of Brassica rapa into Brassica napus canola and inheritance of the B. rapa alleles

Broadening of genetic diversity in spring oilseed Brassica napus L. (AACC, 2n = 38) canola is important for continued improvement of this crop. For this, the vast allelic diversity of the A genome of Brassica rapa L. (AA, 2n = 20) can be utilised. We investigated the prospect of developing canola-quality euploid B. napus lines carrying the alleles of B. rapa from F2 and BC1 (F1 × B. napus) populations of three B. napus × B. rapa interspecific crosses involving one B. napus and three genetically distinct B. rapa parents. In meiosis, the F1 AAC hybrid was expected to show normal segregation for the A genome chromosomes, whereas a range of C chromosomes from zero to nine was expected to be included in the gametes due to random segregation of this haploid set of chromosomes. Subsequent self-pollination, theoretically, should have eliminated the unpaired C chromosomes and resulted in a majority of B. rapa type. However, no B. rapa-type progeny were detected, and all progeny in the F8 conformed to be B. napus type. Correlation between parent and offspring generation, grown in greenhouse or field, was weak to moderate for seed glucosinolate content; however, the simpler genetic control of this trait, involving only the A genome loci, allowed the development of low-glucosinolate lines from this interspecific cross. Of the theoretical number of simple sequence repeat (SSR) marker alleles of B. rapa expected to be present in F4 and F8 populations, about 45% were detected in these populations, suggesting that the loss of these marker alleles occurred prior to the F4 generation. Loss of several SSR loci was also detected in these populations, which probably resulted from homoeologous pairing and rearrangements of the chromosomes of the A and C genomes. Genetic diversity analysis performed on the F8 progeny of two crosses showed that the two populations clustered into distinct groups, which demonstrates that they inherited SSR B. rapa alleles unique to each B. rapa parent. We conclude that B. rapa alleles from diverse sources can be readily incorporated into B. napus progeny by this interspecific crossing method.

Targeted silencing of BjMYB28 transcription factor gene directs development of low glucosinolate lines in oilseed Brassica juncea

Brassica juncea (Indian mustard), a globally important oilseed crop, contains relatively high amount of seed glucosinolates ranging from 80 to 120μmol/g dry weight (DW). One of the major breeding objectives in oilseed Brassicas is to improve the seed-meal quality through the development of low-seed-glucosinolate lines (<30μmol/g DW), as high amounts of certain seed glucosinolates are known to be anti-nutritional and reduce the meal palatability. Here, we report the development of transgenic B.juncea lines having seed glucosinolates as low as 11.26μmol/g DW, through RNAi-based targeted suppression of BjMYB28, a R2R3-MYB transcription factor family gene involved in aliphatic glucosinolate biosynthesis. Targeted silencing of BjMYB28 homologs provided significant reduction in the anti-nutritional aliphatic glucosinolates fractions, without altering the desirable nonaliphatic glucosinolate pool, both in leaves and seeds of transgenic plants. Molecular characterization of single-copy, low glucosinolate homozygous lines confirmed significant down-regulation of BjMYB28 homologs vis-à-vis enhanced accumulation of BjMYB28-specific siRNA pool. Consequently, these low glucosinolate lines also showed significant suppression of genes involved in aliphatic glucosinolate biosynthesis. The low glucosinolate trait was stable in subsequent generations of the transgenic lines with no visible off-target effects on plant growth and development. Various seed quality parameters including fatty acid composition, oil content, protein content and seed weight of the low glucosinolate lines also remained unaltered, when tested under containment conditions in the field. Our results indicate that targeted silencing of a key glucosinolate transcriptional regulator MYB28 has huge potential for reducing the glucosinolates content and improving the seed-meal quality of oilseed Brassica crops.

Costs of glucosinolates in &amp;lt;i&amp;gt;Brassica rapa&amp;lt;/i&amp;gt;: Are they context dependent?

Models predicting optimal levels of plant defense against herbivores typically include two assumptions: 1) defense is both beneficial and costly; and 2) the relationship between costs and benefits of a defense is consistent across environments. However, the expression of costs and benefits of defense may be environmentally dependent. We examined lines of Brassica rapa, previously divergently selected for the defensive trait foliar glucosinolate content. In one set of experiments (Experiment #1), plants were grown in herbivore-free and herbivore-present environments to investigate the costs and benefits of this defense. In a second set of experiments (Experiment #2), plants were grown at two nutrient levels and two temperatures to examine the environmental context of costs of defense. In Experiment #1, increased levels of damage resulted in decreased flower production and plants from high glucosinolate lines received less damage than those from low glucosinolate lines, suggesting a benefit of this defense. In this experiment no cost of defense was detected. In Experiment #2, nutrients had a significant positive effect on flower production at 23°C, but not at 32°C. No significant effects of glucosinolate line nor interaction between nutrient environment and glucosinolate line were detected at 23°C, suggesting that no cost of defense occurred at this lower temperature. Similarly, no significant nutrient environment by glucosenolate line interaction was detected at 32°C. However, a significant effect of glucosinolate line was observed suggesting that at 32°C costs were incurred, but nutrient environment had no mitigating effect. While results from Experiment #1 suggested that defense was beneficial, but not costly, results from Experiment #2 suggested that costs of defense were temperature dependent. For species occupying broad geographic ranges, these findings of temperature-dependent costs are especially insightful with regard to the evolution of defense because differing geographic populations are likely to experience differing temperature environments.

Accumulation of glucosinolate, oil, and erucic acid in developing Brassica seeds

The glucosinolate-free meal from rapeseed can be used as livestock feed, whereas glucosinolate-rich meal can be used as a pesticide. It has also been shown that glucosinolate content in Brassica (rapeseed) seeds can be manipulated by application of chemicals. However, the stage of seed development when potential chemical treatments could be applied has not been clearly identified. Thus, an experiment was conducted to evaluate accumulation of glucosinolate, oil, and erucic acid in developing seeds of rapeseed lines. These included lines that were classified as low or high for glucosinolate content. The florets of eight rapeseed lines were tagged at the 50% flowering stage and were sequentially harvested at 26, 28, 30, 33, 35, 37, 40, 42, 44, 47, 49, and 52 days after flowering (DAF). The oil content in both the high and low-glucosinolate lines increased approximately by a factor of four from 26 to 52 DAF. The oil content accumulation was not affected by glucosinolate content. Erucic acid content in the oil was significantly higher in low glucosinolate lines compared with high glucosinolate lines on 28 and 40 DAF. Developing seeds were most amenable to change in erucic acid content at approximately 37 DAF. The glucosinolate contents in high glucosinolate lines started to increase significantly at 26 DAF and continued up to 33 DAF. However, the glucosinolate content in the low glucosinolate lines increased only from 33 to 35 DAF. This indicates that the greatest accumulation of glucosinolate in developing rapeseed seeds may occur at approximately 26 DAF.

Realized Defense of Artificially Selected Lines of Brassica rapa: Effects of Quantitative Genetic Variation in Foliar Glucosinolate Concentration

Herbivore species-specific responses to a plant secondary compound could result in variable selection on the concentration of that plant chemical. The purpose of this study was to determine if lines of a rapid-cycling Brassica rapa (L.) that had been artificially selected for quantitative differences in foliar glucosinolate content also differed in their realized defense , defined as the number of eggs laid on or level of damage incurred by a plant when exposed to herbivores. I asked whether defense was conferred at the oviposition or feeding stage, whether 2 herbivore species respond similarly to different foliar glucosinolate concentrations, and whether these same 2 species could impose selection in different directions on foliar glucosinolate concentration. I used an oligophagous herbivore, Pieris rapae (L.), and a polyphagous herbivore, Trichoplusia ni Hübner. Feeding was examined for both herbivore species, and oviposition was examined for P. rapae . P. rapae adult females did not favor any single selection line over another as a site for oviposition. However, P. rapae larvae did remove greater amounts of leaf area on plants from lines selected for low glucosinolate content when given a choice. In addition, T. ni larvae caused significantly greater amounts of damage on low glucosinolate line plants. Thus, quantitative variation in glucosinolate content among selected lines does confer differences in realized defense against these 2 herbivores. However, because P. rapae adult females do not appear to discriminate among plants that differ in glucosinolate content, defense is conferred at the level of feeding. Further, in contrast to previous studies, larvae of both the polyphagous and the oligophagous herbivore species responded similarly to quantitative variation in secondary compounds, causing more damage on low glucosinolate lines. This parallel response suggests that both herbivore species should impose selection on defense in the same direction in B. rapa for increased levels of glucosinolate production.

Will low-glucosinolate cultivars of the mustards Brassica juncea and Sinapis alba be vulnerable to insect pests?

Foliar concentrations of the predominant glucosinolates in the mustards B. juncea (allyl glucosinolate, sinigrin) and S. alba (p-hydroxybenzyl glucosinolate, sinalbin) were determined in lines that had been selected in breeding programs for low levels of glucosinolates in their seeds for the oilseed market. The glucosinolate concentrations found in the cotyledons and leaves of the selected lines were also low, often by three or more orders of magnitude, compared with the unselected parent. The flea beetle, Phyllotreta cruciferae Goeze, and the diamondback moth, Plutella xylostella (L.), (both crucifer specialists) fed at equal rates on B. juncea and its low-glucosinolate lines, indicating that these species are insensitive to sinigrin and suggesting that their pest status on low-glucosinolate lines of B. juncea will likely remain unchanged. By contrast, the bertha armyworm, Mamestra configurata Wlk. (a generalist) fed up to fivefold more on the leaves of low-glucosinolate lines, suggesting that its pest status on low-glucosinolate B. juncea is likely to worsen relative to the mustard B. juncea. Flea beetles, diamondback moth, and the bertha armyworm fed at equal rates on the mustard S. alba and a low-glucosinolate line indicating that the status of these pests on low-glucosinolate S. alba will unlikely be much different from the mustard S. alba.Lines of S. alba differing in their sinalbin concentration by 1000-fold had equal levels of antixenosis resistance in their cotyledons against flea beetles, leaving little doubt that this resistance is independent of glucosinolates. Flea beetle resistance was also detected in the cotyledons of a low-glucosinolate line of B. juncea. The resistance was not detected in the parental line or in another low-glucosinolate line and appears to have arisen fortuitously during crossing and selection. Since resistance occurs in a line with concentrations of sinigrin three or more orders of magnitude lower than non-resistant lines, this resistance is also unlikely to be glucosinolate based. The mustards S. alba and B. juncea are a useful source of cotyledon resistance against flea beetles that is compatible with the aims of oilseed breeding. Key words: Glucosinolate, Brassica juncea, Sinapis alba, Phyllotreta cruciferae, Plutella xylostella, Mamestra configurata, resistance, mustard, canola, insect

Molecular markers for low-glucosinolate alleles in oilseed rape (Brassica napus L.)

Two recent studies have mapped QTLs associated with the level of seed glucosinolates in oilseed rape (Brassica napus L.). It was likely that the two most significant QTLs identified in each study were the same, as they were linked to RFLP alleles identified by common DNA probes. To investigate the utility of these probes in breeding programmes, they were used to study RFLPs in a range of low- and high-glucosinolate cultivars and breeding lines. It was shown that all low glucosinolate spring and winter cultivars possessed a specific RFLP fragment identified by probe wg3f7 which is linked to theGSL-1 QTL, and all high-glucosinolate cultivars possessed a specific RFLP fragment identified by probe wg7a8, which is linked to theGSL-2 QTL. Cultivar Ariana, which has intermediate levels of glucosinolates possessed the low-glucosinolate fragment atGSL-1 but the high-glucosinolate fragment atGSL-2. A similar result was found with the cvs. Martina and Bronowski which have intermediate and variable levels of glucosinolates. There were no other RFLP fragments identified by other DNA probes which were specific to either the low- or high-glucosinolate phenotypes. The use of probes wg3f7 and wg7a8 in selection of low-glucosinolate lines in breeding programmes is discussed.

Physical and chemical defences of pods and seeds of white mustard (Sinapis alba L.) against tarnished plant bugs, Lygus lineolaris (Palisot de Beauvois) (Heteroptera: Miridae)

Adult tarnished plant bugs (Lygus lineolaris) fed 5–10 times less frequently on seeds within pods of white mustard (Sinapis alba L.) than on seeds within pods of canola (Barssica napus L.) in no-choice feeding tests in the laboratory. The presence of long, sharp spines (trichomes) on pods of S. alba did not account for all of the difference in feeding on this mustard and on the smooth pods of canola because the feeding rate on shaved pods of S. alba was only twofold higher than on unshaved controls. Lygus lineolaris fed four times less frequently on seeds within pods of a commercial cultivar than on seeds of a near-isogenic low-glucosinolate line of S. alba, suggesting that it is the high concentration of the glucosinolate "sinalbin" in seeds that largely determines the low rate of feeding of L. lineolaris on this species.Plant breeders are currently developing low-glucosinolate lines of mustards such as S. alba for the oilseed market. Seed damage by L. lineolaris in an oilseed S. alba likely will be considerably higher than currently occurs in the mustard S. alba. Key words:Brassica, Sinapis, Lygus, trichome, glucosinolate, feeding damage, antixenosis

Alkenyl Glucosinolate Levels in Androgenic Populations of Brassica napus

AbstractThe levels of individual and total alkenyl glucosinolates in seeds of microspore‐derived spontaneous diploid plants from low by high and low by low glucosinolate parent crosses were examined to assess the utility of haploidy in canola breeding. The distributions of lines in the populations supported previous proposals that alkenyl glucosinolate levels are under multigenic control. Levels of all of the individual glucosinolates were positively correlated and were significantly reduced in canola‐quality material in comparison to rapeseed‐quality material. The populations of microspore‐derived lines from low × high crosses were skewed to high glucosinolate levels but the population from a low × low glucosinolate cross had a greater proportion of low glucosinolate lines. The former observations can be explained in terms of the dominance of genes for high glucosinolate levels in Brassica napus. The present findings contradict previous reports that androgenic lines have higher glucosinolate content than the parents and in fact, haploidy may select for low glucosinolate lines when crosses between low glucosinolate parents are used.

GLUCOSINOLATE CONTENT OF MATURING PODS AND SEEDS OF HIGH AND LOW GLUCOSINOLATE SUMMER RAPE

3-Butenyl-, 4-pentenyl-, 2-hydroxy-3-butenyl- and 4-hydroxy-3-indolylmethylglucosinolate were found to be major glucosinolates in developing pod tissue and seed of both high and low glucosinolate lines of rapeseed. These glucosinolate profiles along with decreases in the glucosinolate content of pod tissue over a period of 18 d after pollination, and increases in the seed, were consistent with the pod tissue being a source of glucosinolate accumulating in the seed.Key words: Rapeseed, Brassica napus L., Cruciferae, glucosinolate, seed development

Glucosinolate profiles of international rapeseed lines (Brassica napus and Brassica campestris)

AbstractSeed samples from a range of international rapeseed lines (Brassica napus L and B campestris L) were analysed for glucosinolate content using high performance liquid chromatography (HPLC). This analytical method provided a total profile consisting of eight different glucosinolates, including those used to define the Canola standard. The data indicated that broad categories of distinct glucosinolate profiles existed among the seed lines analysed. Indole glucosinolate content, expressed as a percentage of total glucosinolate content, was higher in the low glucosinolate lines of B napus. The actual contents of all eight glucosinolates in the seed were variable, particularly gluconapin, glucobrassicanapin and progoitrin. The importance of this HPLC procedure in facilitating selection for high or low contents of individual glucosinolates is discussed.