1BioMarka, JCCI, Institute of Land and Food Resources, University of Melbourne, Vic. 3010.
2JCCI, Victorian Institute for Dryad Agriculture, Department of Primary Industries, Private Bag 260, Horsham, Vic 3401.
Boron is a micronutrient essential for the growth of vascular plants, but may also result in phytotoxicity when present in excessive amounts. In general, tissue boron concentration greater than 300 mg/kg dry weight may result in toxicity. Nineteen Brassica rapa genotypes were screened for tolerance with differing boron concentrations in hydroponic and soil bioassays. The most tolerant and susceptible genotypes were selected and assessed for boron tissue concentrations. The tolerant genotypes contained significantly lower boron concentrations than the susceptible genotypes and the bulk soil, indicating that tolerance is mediated by an exclusion mechanism. Genetic diversity analysis with sequence related amplified polymorphism (SRAP) molecular markers revealed sufficient diversity among tolerant and susceptible genotypes for genome mapping of the tolerance trait.
Boron tolerant Brassica rapa genotypes were identified by hydroponic and soil assays. SRAP markers identified genetic differences and tissue boron levels indicate that tolerant lines exclude boron .
Boron tolerance, exclusion, SRAP markers, diversity
The Australian canola industry has expanded dramatically since the 1990’s due to the availability of better adapted lines and the development of improved agronomic practices. However, there are several major factors limiting canola production such as susceptibility to blackleg, pod dehiscence and manganese (Mn) and boron (B) toxicity.
Boron is absorbed by plants from the soil solution as undissociated boric acid (pKa = 9.25, 25°C; Dordas and Brown 2000). The mechanism of B uptake remains a controversial subject and there is evidence supporting both active and passive uptake into plant cells. However, passive uptake is the most widely accepted mechanism for higher plants (Nable et al 1990; Nable et al 1997) particularly at high concentrations in the soil solution.
When present in tissues at concentrations greater than 300 mg/kg dry weight, B is phytotoxic to plant growth. The most obvious symptoms of toxicity are leaf burns, chlorotic and necrotic patches, often visible at the margins and tips of older leaves. These symptoms reflect the tissue distribution of B in most species, with accumulation at the ends of transpiration streams (Nable et al 1990) Because the range between deficient and toxic amounts of soil B is narrow, the potential for either inadequate or excessive soil B availability is great. In particular, the calcarasols that are present across much of the Wimmera and Mallee regions of southern Australia are high in salt, exchangeable sodium and available B (Nuttall et al 2003). These conditions limit the ability of crops to take up water from deep (>60cm) in the soil profile. Treating the soil to remove or reduce the B concentration is not economically feasible. However, selecting or breeding crop cultivars with high B tolerance is an effective approach to increase yields on high B soils.
In order to breed for tolerance, the genetics of the trait and its genetic diversity within the germplasm need to be understood. For this, a cross between a genetically diverse susceptible and tolerant genotype is required to produce a genome map. Linkage maps have previously been constructed to identify the chromosomal regions involved in B tolerance in barley, wheat and Brassica species (Hu et al 1995; Jefferies et al 2000; Xu et al 2001). A robust and reproducible bioassay has been developed to accurately discriminate among wheat genotypes that were susceptible or tolerant to B (Chantachume et al 1995). Recently a DNA-based marker protocol, called sequence related amplified polymorphism (SRAP) analysis was developed and employed for studying the genetic diversity within several Brassica species. The SRAP technique specifically targets coding sequences and results in the screening of co-dominant markers (Quiros and Li 2001).
This report details the selection of genetically diverse susceptible and tolerant B. rapa genotypes that will be used in studies to determine the genetics of B tolerance. Brassica rapa shares a common genome with the oilseed types B. napus and B. juncea, so we chose to investigate this character in the diploid rather than the allotetraploid species.
Hydroponic and soil bioassays were undertaken to identify susceptible and tolerant B. rapa genotypes. For the hydroponic assay, nineteen B. rapa genotypes (wwy sarson, PI212083, Bharatar-1, KatmanduCL-10, Nagoraka, CPI156420, Mex H, Jap Local, Local, SV70/8368, Shillong, Kaga, PI324507, Great Luck, ConFooC, Mex J, Candle, PI175608 and Span) were screened against seven concentrations of B (0, 25, 50, 75, 100, 125 and 150 μM). The basal hydroponic solution comprised 500 μM Ca(NO3)2.4H2O, 2.5 μM ZnSO4.7H2O and 15 μM H3BO3 with added concentrations of B as boric acid. Seeds were surface sterilized in 70% ethanol and 0.5% NaOCl and pre-germinated on moist filter paper under sterile conditions. Six seedlings with a uniform root length for each genotype per treatment were transferred into polystyrene holders for growth in different concentrations of B. Two experiments with six replications within each treatment were carried out and primary root length was measured for each seedling after 15 d in hydroponic solution.
The two most susceptible (Shillong and Kaga) and tolerant (wwy sarson and Local) B. rapa genotypes were screened in a pot trial. Six seeds of each genotype were sown directly into plastic tubs filled with 12 kg of soil taken from a typical calcarasol site in the Birchip region (-35.80oS, 142.87oE) with a basal soil B level of 4 mg/kg (hot water soluble). Additional B was added to the soil as boric acid to the equivalent final concentrations of 25 and 50 mg/kg of hot water soluble B. Two replications of each treatment were carried out and plants were harvested at five weeks (five to six leaf stage) after sowing. Total above-ground tissue was dried at 450C for 48 h and analysed for B concentration using Inductive Coupled Plasma Spectrometry (ICP) analysis. For this, samples were digested overnight with concentrated nitric acid, incubated for 3 h at 90 0C and a 1/6.7 dilution of the extract was analysed.
SRAP primers were screened against all of the B. rapa genotypes by polymerase chain reaction (PCR) in a PTC200 machine (MJ Research, USA) using the following reaction conditions: the final reaction volume of 25 μl comprised PCR buffer (Invitrogen, USA), 3mM MgCl2, 0.24 mM each dNTPs (Invitrogen, USA), 0.24 μM of forward and reverse primer, 1 unit of Taq polymerase (Invitrogen, USA) and 40 ng of template DNA. The primers and PCR cycle were as described by Quiros and Li (2001). Amplification products were resolved on 1.4% agarose gels in Tris-borate EDTA (TBE) buffer and stained with ethidium bromide. Products were scored as either present (1) or absent (0) and a binary matrix of pair-wise genetic distance was constructed using Nei’s unbiased measures of genetic distance (Nei, 1978). A dendrogram, showing the genetic relationships among accessions, was constructed from the pair-wise genetic distance values based on the un-weighted pair-group method using arithmetic means (UPGMA) analysis in the Molecular Evolutionary Genetic Analysis (MEGA) software version 2.1 (Kumar et al. 2001).
The hydroponic screening identified the two most susceptible genotypes were (Shillong and Kaga) and the two most tolerant genotypes (wwy sarson and Local). Compared to the control treatment (0 μM B), the susceptible genotypes had a reduced root length in all B concentration, with a maximum of 60% reduction in 150 μM B. Conversely, the tolerant genotypes did not show reduction in root length at concentrations less than 125 μM B and were only reduced by ~15% at 150 μM B (Fig. 1). After 15 days in the 150 μM B hydroponic solution, the sensitive genotypes showed severe symptoms of B toxicity such as chlorotic and necrotic patches, particularly at the leaf tips and margins.
Figure 1. Effect of B concentration on primary root length.
Following the 50 mg/kg soil treatment, the dry weight of wwy sarson was reduced by <10% and the dry weights of Shillong and Kaga were reduced by 75.3% and 75% respectively. Surprisingly, the dry weight of Local was also substantially reduced (52.6%) although no B toxicity symptoms were observed. ICP analysis detected a much lower amount of B in the tissues of wwy sarson and Local than the tissues of Shillong and Kaga at 50 mg/kg soil B. This could indicate that wwy sarson and Local genotypes exclude B and that Shillong and Kaga absorb (either passively or actively) from the environment through their roots.
Figure 2. Tissue B levels of four B. rapa genotypes under three levels of soil B (A=control, B=25 mg/kg, C=50 mg/kg)
From a total of 15 SRAP primer combinations screened, 14 were polymorphic and generated 128 scorable markers for assessing the diversity among B. rapa genotypes. The pair-wise matrix revealed 44% diversity between the susceptible genotype Kaga and tolerant genotype wwy sarson. The genetic relationship among all of the B. rapa genotypes was demonstrated by the dendrogram (Fig. 3).
Figure 3. Genetic relationship among B. rapa genotypes using SRAP markers
Due to the distinct differences in tolerance to B in both hydroponic and soil assay and the genetic diversity detected, the genotypes Kaga and wwy sarson represent suitable parents for the production of a hybrid population for the future genome mapping of B tolerance.
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