Introgression of B-genome chromosomes in a doubled haploid population of Brassica napus x B. carinata.

The Brassica B-genome species possess many valuable agronomic and disease resistance traits. To transfer traits from the B genome of B. carinata into B. napus, an interspecific cross between B. napus and B. carinata was performed and a doubled haploid (DH) population was generated from the BC2S3 generation. Successful production of interspecific DH lines as identified using B-genome microsatellite markers is reported. Five percent of DH lines carry either intact B-genome chromosomes or chromosomes that have deletions. All of the DH lines have linkage group J13/B7 in common. This was further confirmed using B. nigra genomic DNA in a fluorescent in situ hybridization assay where the B-genome chromosomes were visualized and distinguished from the A- and C-genome chromosomes. The 60 DH lines were also evaluated for morphological traits in the field for two seasons and were tested for resistance to blackleg, caused by Leptosphaeria maculans, under greenhouse conditions. Variation in the DH population followed a normal distribution for several agronomic traits and response to blackleg. The lines with B-genome chromosomes were significantly different (p < 0.01) from the lines without B-genome chromosomes for both morphological and seed quality traits such as days to flowering, days to maturity, and erucic acid content.

Identification of quantitative trait loci (QTL) for oil and protein contents and their relationships with other seed quality traits in Brassica juncea.

A detailed RFLP-genomic map was used to study the genetics of oil, seed and meal protein and sum of oil and seed/meal protein contents in a recombinant doubled-haploid population developed by crossing black- and yellow-seeded Brassica juncea lines. Two yellow seed color genes (SC-B4, SC-A6) and one QTL for erucic acid content (E(1b)) showed pleiotropic effect for oil, protein and sum of oil and seed/meal protein contents. Six (O-A1, O-A6, O-A9, O-B3, O-B4, O-B5) and five (SP-A1, SP-A9, SP-B4, SP-B6, SP-C) QTLs were significant for oil and seed protein contents, respectively. Tight linkage of three of these QTLs (SP-A1, SP-A9, SP-B4, O-A1, O-A9, O-B4), with opposite effects, poses challenge to the plant breeders for simultaneous improvement of negatively correlated (r = -0.7**) oil and seed protein contents. However, one QTL for oil content (O-B3) and two for seed protein content (SP-B6, SP-C) were found to be unlinked, which offer the possibility for simultaneous improvement of these two traits. QTLs significant for meal protein (MP-A1, MP-A6, MP-A9, MP-B5, MP-B6) were significant at least for oil, seed protein or sum of oil and seed/meal protein contents (T-A6, T-A7, T-B4, T-B5). Sum of oil and seed protein contents and sum of oil and meal protein contents had a perfect correlation, as well as same epistatic interactions and QTLs with similar additive effect. This indicates that protein in seed or meal has practically the same meaning for breeding purposes. Epistatic interactions were significant for the quality traits, and their linkage reflected association among the traits.

Mapping genes for resistance to Leptosphaeria maculans in Brassica juncea.

Blackleg disease of crucifers, caused by the fungus Leptosphaeria maculans, is a major concern to oilseed rape producers worldwide. Brassica species containing the B genome have high levels of resistance to blackleg. Brassica juncea F2 and first-backcross (B1) populations segregating for resistance to a PG2 isolate of L. maculans were created. Segregation for resistance to L. maculans in these populations suggested that resistance was controlled by two independent genes, one dominant and one recessive in nature. A map of the B. juncea genome was constructed using segregation in the F2 population of a combination of restriction fragment length polymorphism (RFLP) and microsatel lite markers. The B. juncea map consisted of 325 loci and was aligned with previous maps of the Brassica A and B genomes. The gene controlling dominant resistance to L. maculans was positioned on linkage group J13 based on segregation for resistance in the F2 population. This position was confirmed in the B1 population in which the resistance gene was definitively mapped in the interval flanked by pN199RV and sB31143F. The provisional location of the recessive gene controlling resistance to L. maculans on linkage group J18 was identified using a subset of informative F2 individuals.

Molecular markers for seed colour in Brassica juncea.

A detailed RFLP map was used to map QTLs associated with seed colour in Brassica juncea using a doubled-haploid population derived from a cross between a black/brown-seeded cultivar and a yellow-seeded breeding line. Segregation analysis suggested that seed colour was under control of 2 unlinked loci with duplicate gene action. However, QTL analysis revealed 3 QTLs, SC-B4, SC-A10 and SC-A6, affecting seed colour. The QTLs were consistent across environments, and individually explained 43%, 31%, and 16%, respectively, and collectively 62% of the phenotypic variation in the population. Digenic interaction analysis showed that closest flanking locus of QTL SC-B4, wg7b6cNM, had strong epistasis with the locus wg5a1a, which is tightly linked to QTL SC-A6. The interaction of these 2 loci explained 27% of the phenotypic variation in the population, while the whole model explained 84%. In a multiple regression model, the effects of QTL SC-A10, as well as its interaction with other loci, were non-significant, whereas the effects of loci wg7b6cNM and wg5a1a and their interaction were significant. Ninety-eight percent of the DH lines carried the expected alleles of loci wg7b6cNM and wg5a1a for seed colour, confirming that only these 2 loci were linked to seed colour in B. juncea. Four additional digenic interactions significantly affected seed colour, and all 5 digenic interactions were consistent across environments.

Molecular mapping of seed aliphatic glucosinolates in Brassica juncea.

An RFLP genomic map with 316 loci was used to study the inheritance of aliphatic glucosinolates in Brassica juncea using doubled-haploid (DH) populations developed from a cross between RLM-514, an agronomically superior non-canola quality B. juncea (high erucic acid and high glucosinolates), and an agronomically poor canola quality B. juncea breeding line. Two QTLs (GSL-A2a and GSL-A2b) associated with 3-butenyl were consistent across years and locations, and explained 75% of the phenotypic variance in the population. Three QTLs (GSL-A2a, GSL-F, GSL-B3) affected 2-propenyl and explained 78% of the phenotypic variance in the population. For total aliphatic glucosinolates, five QTLs explained 30% to 45% of the total phenotypic variance in the population in different environments. Several QTLs (GSL-A7 and GSL-A3) were highly inconsistent in different environments. Major QTLs (GSL-A2a and GSL-A2b) associated with individual glucosinolates were non-significant for total aliphatic glucosinolates. A marker-assisted selection strategy based on QTLs associated with individual glucosinolates rather than total aliphatic glucosinolates is proposed for B. juncea.

RFLP linkage analysis and mapping genes controlling the fatty acid profile of Brassica juncea using reciprocal DH populations.

An RFLP linkage map, comprising 300 linked and 16 unlinked loci, was constructed using reciprocal DH populations of Brassica juncea. The linked loci were organized into 18 linkage groups and seven unlinked segments, covering a total map distance of 1,564 cM. The A and B genomes were identified. The chi(2) test showed that 96.1% of the common intervals in the two populations differed non-significantly for recombination fractions, thus strongly suggesting the absence of sex-based differences for recombination fractions in B. juncea. Two QTLs, E(1a) and E(1b), significantly affected erucic acid content, and individually explained 53.7% and 32.1%, respectively, and collectively 85.8% of the phenotypic variation in the population. The QTLs E(1a) and E(1b) showed epistasis, and the full model including epistasis explained nearly all of the phenotypic variation in the population. The QTLs E(1a) and E(1b) were also associated with contents of oleic, linoleic and linolenic acids. Three additional QTLs (LN(2), LN(3) and LN(4)) significantly influenced linolenic acid content. The QTL LN(2) accounted for 35.4% of the phenotypic variation in the population. Epistatic interactions were observed between the QTLs E1a and LN(2). The stability of the detected QTLs across years and locations, and breeding strategies for improving the fatty acid profile of B. juncea, are discussed.

Vacuolar H+-ATPase, but not mitochondrial F1F0-ATPase, is required for aluminum resistance in Saccharomyces cerevisiae.

It was recently shown that vacuolar ATPase and mitochondrial F1F0-ATPase activities are induced by aluminum (Al) in an Al-resistant cultivar of wheat, suggesting that induction of these enzymes could be an adaptive trait involved in Al resistance. To test this hypothesis, we used the Saccharomyces cerevisiae model system. In yeast, unlike wheat, the activity, transcript and protein levels of mitochondrial F1F0-ATPase, but not vacuolar ATPase, are induced by Al, while plasma membrane P-ATPase activity is inhibited. However, yeast vacuolar ATPase mutant strains are hypersensitive to Al, while F1F0-ATPase mutant strains exhibit wild-type growth. These data suggest that vacuolar ATPase activity is involved in Al resistance, with ATP required for this activity supplied by mitochondrial F1F0-ATPase or fermentation.

Induction of vacuolar ATPase and mitochondrial ATP synthase by aluminum in an aluminum-resistant cultivar of wheat.

Two 51-kD aluminum (Al)-induced proteins (RMP51, root membrane proteins of 51 kD) were recently discovered in an aluminum-resistant cultivar of wheat (Triticum aestivum) cv PT741 (Basu et al., 1994a). These proteins segregate with the aluminum resistance phenotype in a segregating population arising from a cross between Al-resistant cv PT741 and Al-sensitive cv Katepwa (Taylor et al., 1997). The proteins have been purified by continuous elution electrophoresis and analyzed by peptide microsequencing. Sequence analysis of the purified peptides revealed that they are homologous to the B subunit of the vacuolar H+-ATPase (V-ATPase) and the alpha- and beta-subunits of the mitochondrial ATP synthase (F1F0-ATPase). To confirm that these ATPases are induced by Al, ATPase activity and transcript levels were analyzed under Al stress. Both V-ATPase and F1F0-ATPase activities were induced by Al and responded in a dose-dependent manner to 0 to 150 microM Al. In contrast, plasma membrane H+-ATPase (P-ATPase) activity decreased to 0.5x control levels, even when plants were exposed to 25 microM Al. Northern analysis showed that the transcript encoding the B subunit of V-ATPase increased by 2.2x in a dose-dependent manner, whereas levels of the transcript encoding the alpha-subunit of F1F0-ATPase remained constant. The effect of Al on ATPase activity in other cultivars was also examined. The Al-resistant cultivar, cv PT741, was the only cultivar to show induction of V- and F1F0-ATPases. These results suggest that the V-ATPase in cv PT741 is responding specifically to Al stress with the ATP required for its activity supplied by ATP synthase to maintain energy balance within the cell.

Molecular strategies for improving waterlogging tolerance in plants.

Plants, like animals, are obligate aerobes, but due to their inability to move, have evolved adaptation mechanisms that enable them to survive short periods of low oxygen supply, such as those occurring after heavy rain or flooding. Crop plants are often grown on soils subject to waterlogging and many are sensitive to waterlogging of the root zone. The combination of unfavourable weather conditions and suboptimal soil and irrigation techniques can result in severe yield losses. The molecular basis of the adaptation to transient low oxygen conditions has not been completely characterized, but progress has been made towards identifying genes and gene products induced during low oxygen conditions. Promoter elements and transcription factors involved in the regulation of anaerobically induced genes have been characterized. In this paper an account is presented of the molecular strategies that have been used in an attempt to increase flooding tolerance of crop plants.

Arabidopsis thaliana: a source of candidate disease-resistance genes for Brassica napus.

Common structural and amino acid motifs among cloned plant disease-resistance genes (R genes), have made it possible to identify putative disease-resistance sequences based on DNA sequence identity. Mapping of such R-gene homologues will identify candidate disease-resistance loci to expedite map-based cloning strategies in complex crop genomes. Arabidopsis thaliana expressed sequence tags (ESTs) with homology to cloned plant R genes (R-ESTs), were mapped in both A. thaliana and Brassica napus to identify candidate R-gene loci and investigate intergenomic collinearity. Brassica R-gene homologous sequences were also mapped in B. napus. In total, 103 R-EST loci and 36 Brassica R-gene homologous loci were positioned on the N-fo-61-9 B. napus genetic map, and 48 R-EST loci positioned on the Columbia x Landsberg A. thaliana map. The mapped loci identified collinear regions between Arabidopsis and Brassica which had been observed in previous comparative mapping studies; the detection of syntenic genomic regions indicated that there was no apparent rapid divergence of the identified genomic regions housing the R-EST loci.

Evolution of a functionally related lactate dehydrogenase and pyruvate decarboxylase pseudogene complex in maize.

A large proportion of the maize genome is repetitive DNA (60-80%) with retrotransposons contributing significantly to the repetitive DNA component. The majority of retrotransposon DNA is located in intergenic regions and is organized in a nested fashion. Analysis of an 8.2-kb segment of maize genomic DNA demonstrated the presence of three retrotransposons of different reiteration classes in addition to lactate dehydrogenase and pyruvate decarboxylase pseudogenes. Both of the pseudogenes were located within a defective retrotransposon element (LP-like element) which possessed identical long terminal repeats (LTRs) with inverted repeats at each end, a primer binding site, a polypurine tract, and generated a 5-bp target site duplication. A model describing the events leading to the formation of the LP-like element is proposed.

Molecular mapping of resistance to Leptosphaeria maculans in Australian cultivars of Brassica napus.

Doubled haploid (DH) lines together with a cotyledon bioassay were employed for the molecular analysis of resistance to the blackleg fungus Leptosphaeria maculans in the Australian Brassica napus cultivars Shiralee and Maluka. We used bulked segregant analysis to identify 13 RAPD and two RFLP markers linked to the resistance phenotype and mapped these markers in the segregating DH population. Our data suggest the presence of a single major locus controlling resistance in the cultivar Shiralee, confirming our previous results obtained from Mendelian genetic analyses. In addition, preliminary mapping data for the cultivar Maluka also support a single locus model for resistance and indicate that the resistance genes from 'Shiralee' and 'Maluka' are either linked or possibly identical. The molecular markers identified in this study should be a useful tool for breeding blackleg resistant varieties using marker-assisted selection, and are the essential first step towards the map-based cloning of this resistance gene.

Characterization of Hypoxically Inducible Lactate Dehydrogenase in Maize.

Oxygen deprivation induces a wide variety of genes, but the most extensively studied are those encoding enzymes of the glycolytic pathway. Lactate dehydrogenase (LDH, EC 1.1.1.27) activity increases up to 3.5-fold in maize (Zea mays L.) roots during several days of hypoxic induction. This increase in activity is accompanied by a decrease in in vitro enzyme stability. LDH activity in aerobic root extracts has an in vitro half-life of 240 min, decreasing to 100 min in 72-h hypoxically induced plant root extracts. The increase in enzyme activity during hypoxic induction is the result of increased protein levels, which correlate with increased transcript levels. Two ldh transcripts of 1.3 and 1.7 kb are induced, with maximum levels reached by 8 and 24 h, respectively. This suggests that the two ldh genes are differentially regulated. Treatment with the protein synthesis inhibitor cycloheximide does not preclude ldh induction during the first few hours of hypoxic stress, suggesting that new protein synthesis may not be essential for elevation of ldh transcript levels under hypoxic conditions. The rapid and substantial increase in ldh mRNA levels under hypoxic conditions and in the presence of cycloheximide suggests that the ldh gene may be valuable in analyzing the hypoxic signal transduction pathway.

Molecular cloning and expression of a turgor-responsive gene in Brassica napus.

During droughting plants activate a number of genes involved in adaptation to water stress. We have isolated one such gene, btg-26, from Brassica napus. Expression of btg-26 is induced in leaf tissue within 72 h of withholding water. At 81% relative water content (RWC), when the plant is just beginning to show signs of wilting, expression is already increased six-fold over levels found in leaf tissue from fully hydrated plants. btg-26 expression reaches a maximum eleven-fold induction at 63% RWC, then transcript levels decrease as RWC continues to drop. btg-26 is also activated in plants exposed to high salinity, low temperature, heat shock and the plant hormone abscisic acid. Analysis of the deduced amino acid sequence revealed similarity to the dehydrogenase family of enzymes. These results suggest that btg-26 encodes a protein whose function may be required early during general osmotic stress in some unknown adaptive metabolic pathway.

Hypoxically inducible barley alanine aminotransferase: cDNA cloning and expression analysis.

A 1.75 kb cDNA containing the entire coding sequence of the hypoxically inducible alanine aminotransferase (AlaAT) from barley roots was isolated and sequenced. This clone has an open reading frame of 1446 bp, and a deduced amino acid sequence of 482 residues, giving an estimated protein molecular mass of 52,885 Da. RNA blot analysis of barley root tissue showed a 4-fold increase of a single AlaAT-2 mRNA band after 12-24 hours of hypoxic stress, followed by a decrease in message levels after 48 h of hypoxic conditions. AlaAT-2 protein concentration increased in a similar pattern to AlaAT activity in root tissue, to almost 6-fold the aerobic level after 96 h of hypoxic stress. AlaAT-2 activity increased more than 2-fold in roots of Panicum miliaceum exposed to hypoxia, and is the same isoform as the light inducible AlaAT in P. miliaceum leaves. The unique expression patterns of AlaAT-2 in root and leaf tissue upon exposure to different environmental stimuli is also discussed.

Long-Term Anaerobic Metabolism in Root Tissue (Metabolic Products of Pyruvate Metabolism).

The onset of anaerobiosis in barley root tissue (Hordeum vulgare L. cv Himalaya) results in the following metabolic responses. There are rapid increases in the levels of pyruvate, lactate, and ethanol. Malate and succinate concentrations increase over the first 12 h, after which they return to the levels found in oxygenated root tissue. Alanine concentration increases over the first 12 h, and this is matched by a corresponding decrease in aspartate. The initial stoichiometric decline in aspartate and increase in alanine suggests that the amino group of aspartate is conserved by transaminating pyruvate to alanine. Aspartate catabolism also probably provides the initial source of carbon for reduction to succinate under anoxic conditions. Under long-term anaerobiosis (>24 h), there is no further accumulation of any of the fermentative end products other than ethanol, which also represents the major metabolic end product during long-term anaerobiosis. Although a number of the enzymes involved in fermentative respiration have been found to be induced under anaerobic conditions, neither aspartate amino-transferase nor malate dehydrogenase is induced in barley root tissue. The observations suggest that the long-term adaptations to hypoxic conditions may be quite different than the more well-characterized short-term adaptations.

Purification and characterization of an anaerobically induced alanine aminotransferase from barley roots.

Alanine aminotransferase (AlaAT, EC 2.6.1.2) is an enzyme that is induced under anaerobic conditions in cereal roots. In barley (Hordeum vulgare L.) roots, there are a number of isoforms of AlaAT. We have identified the anaerobically induced isoform and have purified it to homogeneity. The isolation procedure involved a two-step ammonium sulfate precipitation, gel filtration, ion-exchange chromatography, and chromatofocusing. The enzyme was purified approximately 350-fold to a specific activity of 2231 units/milligram protein. The apparent molecular masses of the native and sodium dodecyl sulfate-denatured AlaAT proteins are 97 and 50 kilodaltons, respectively, indicating that the native enzyme is probably a homodimer. AlaAT has a number of interesting characteristics when compared with other plant aminotransferases. AlaAT does not require the presence of pyridoxyl-5-phosphate to retain its activity, and it appears to be very specific in the reactions that it will catalyze.

Identification and characterization of a hypoxically induced maize lactate dehydrogenase gene.

In cereal root tissue, hypoxia induces the enzyme lactate dehydrogenase (LDH); (S)-lactate:NADH oxidoreductase, EC 1.1.1.27). In barley, both biochemical and genetic data indicate that five isozymes are induced under hypoxia. These isozymes are tetramers and arise from the random association of the products of two Ldh genes. The induction of LDH activity in root tissue has been shown to be correlated to an increase in LDH protein and Ldh mRNA. In order to more fully characterize the hypoxic induction of LDH, we have isolated a maize Ldh genomic clone which has strong homology at both the amino acid and nucleotide level to the barley LDH cDNA clones. The Ldh1 gene consists of two exons separated by a 296 bp intron, has the expected eukaryotic regulatory signals and a sequence that has strong homology to the maize anaerobic regulatory element.

Anaerobic induction of alanine aminotransferase in barley root tissue.

Alanine aminotransferase, otherwise called glutamate-pyruvate aminotransferase (GPT), activity increases up to fourfold during several days of anaerobic induction in barley (Hordeum vulgare L.) roots, reaching a maximum activity of 13 international units per gram fresh weight. This increase in activity paralleled the increase in alcohol dehydrogenase activity in the same root tissue. Upon return to aerobic conditions, the induced GPT activity declined with an apparent half-life of 2 days. The isozyme profile of GPT in barley root tissue comprised one band of activity; in maize there were three bands of activity, the bands with greater mobility had much lower activity. Native polyacrylamide gel electrophoresis indicated that the induction of GPT activity results from an increase in the level of activity of these bands; no other activities were detected. When root tissue was induced under different levels of hypoxia (0%, 2%, 5%, and 21% O(2)), changes in GPT activity were found to increase with lower levels of oxygen. Comparisons of GPT induction in barley, maize (Zea mays), rye, (Secale cereale) and wheat (Triticum aestivum) indicate that this enzyme is induced in the root tissue of all of these cereals; however, anaerobic root conditions do not result in the induction of GPT activity in leaf tissue. The dependence of GPT induction on high levels of nitrate in the media was tested by comparing activity levels in Hoagland solution and a nitrate-free nutrient solution. GPT activity was induced to similar levels under both conditions. These results indicate that alanine aminotransferase shows a very similar pattern of induction to alcohol dehydrogenase in barley root tissue and may be important in anaerobic glycolysis.

Induction of alcohol dehydrogenase and lactate dehydrogenase in hypoxically induced barley.

In barley (Hordeum vulgare L.), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH) are induced by anaerobiosis in both aleurone layers and roots. Under aerobic conditions, developing seeds of cv Himalaya accumulate ADH activity, which survives seed drying and rehydration. This activity consists almost entirely of the ADH1 homodimer. Activity of LDH also increases during seed development, but the level of activity in dry or rehydrated seeds is very low, indicating that this enzyme may not be involved in anaerobic glycolysis during the initial stages of germination. In contrast to ADH, the LDH isozymes present in developing seeds are similar to those found in uninduced and induced roots. Developmental expression of ADH and LDH was monitored from 0 to 24 days postgermination. Neither activity was induced to any extent in the germinating seeds; however, both enzymes were highly induced by anoxia in root tissue during development. Based on gel electrophoresis, this increase in activity results from the differential expression of different Adh and Ldh genes in root tissue. The changes in ADH and LDH activity levels were matched by changes in the amount of these particular proteins, indicating that the increase in activity results from de novo synthesis of these two proteins. The level of inducible LDH activity in an ADH1(-) mutant was not found to differ from cv Himalaya. We suggest that although the ADH(-) plants are more susceptible to flooding, they are not capable of responding to the lack of ADH1 activity by increasing the amount of LDH activity in root tissue.