JXB Advance Access originally published online on May 21, 2004
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Journal of Experimental Botany, Vol. 55, No. 401, pp. 1335-1341, June 1, 2004
© 2004 Oxford University Press
RESEARCH PAPER |
Molecular mapping of a gene responsible for Al-activated secretion of citrate in barley
Received 14 January 2004; Accepted 16 March 2004
1 Faculty of Agriculture, Kagawa University, Ikenobe 2393, Miki-cho, Kita-gun, Kagawa 761-0795, Japan
2 Research Institute for Bioresources, Okayama University, Chuo 2-20-1, Kurashiki 710-0046, Japan
* To whom correspondence should be addressed. Fax: +81 87 891 3137. E-mail: maj{at}ag.kagawa-u.ac.jp
Abbreviations: QTL, quantitative trait locus; RFLP, restriction fragment length polymorphism.
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Abstract |
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Aluminium (Al) toxicity is an important limitation to barley (Hordeum vulgare L.) on acid soil. Al-resistant cultivars of barley detoxify Al externally by secreting citrate from the roots. To link the genetics and physiology of Al resistance in barley, genes controlling Al resistance and Al-activated secretion of citrate were mapped. An analysis of Al-induced root growth inhibition from 100 F2 seedlings derived from an Al-resistant cultivar (Murasakimochi) and an Al-sensitive cultivar (Morex) showed that a gene associated with Al resistance is localized on chromosome 4H, tightly linked to microsatellite marker Bmag353. Quantitative trait locus (QTL) analysis from 59 F4 seedlings derived from an F3 plant heterozygous at the region of Al resistance on chromosome 4H showed that a gene responsible for the Al-activated secretion of citrate was also tightly linked to microsatellite marker Bmag353. This QTL explained more than 50% of the phenotypic variation in citrate secretion in this population. These results indicate that the gene controlling Al resistance on barley chromosome 4H is identical to that for Al-activated secretion of citrate and that the secretion of citrate is one of the mechanisms of Al resistance in barley. The identification of the microsatellite marker associated with both Al resistance and citrate secretion provides a valuable tool for marker-assisted selection of Al-resistant lines.
Key words: Aluminium, barley, citrate secretion, gene mapping, microsatellite marker, resistance.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Aluminium (Al) resistance differs greatly among plant species and barley is the most sensitive of the small grain crops (Foy, 1983). Al toxicity limits the growth and productivity of barley on acid soils and the expansion of barley as a crop into many agricultural areas of the world (Minella and Sorrells, 1992). Soil is limed in some areas to improve barley growth and productivity on acid soils, but this practice is often economically unfeasible (Alva et al., 1986). Furthermore, surface application of lime cannot alleviate toxic subsoil Al, which presents a barrier to deep rooting and the uptake of water and nutrients.
An alternative strategy for improving barley productivity on acid soils is to select for cultivars with increased Al resistance or to breed Al-resistant cultivars by introducing Al-resistant genes. Both the genetic and physiological mechanisms of Al resistance in barley need to be elucidated to achieve this purpose. A wide genetic variation in Al resistance has been reported in barley (Reid et al., 1971; Minella and Sorrells, 1992; Ma et al., 1997a). Genetic analysis indicates that Al resistance in barley is controlled by a single dominant gene (Alp) (Reid et al., 1971; Minella and Sorrells, 1992; Raman et al., 2002). Trisomic analysis revealed that the gene conferring Al resistance from the Al-resistant variety Dayton was located on chromosome 4H (Minella and Sorrells, 1997). The gene was further mapped to the long arm of chromosome 4H, 2.1 cM proximal to the marker Xbcd1117 and 2.1 cM distal to the markers Xwg464 and Xcdo1395, by using an F2 population of Dayton (highly resistant)/Harlan Hybrid (moderately sensitive) with restriction fragment length polymorphism (RFLP) markers (Tang et al., 2000). Recently, Raman et al. (2002) used a different F2 progeny derived from a single cross between Yambla (moderately Al-resistant) and WB229 (Al-resistant) and also mapped the Al-resistant gene (Alt) on chromosome 4H. They further found that four chromosome 4H-specific microsatellite markers (Bmac310, Bmag353, HVM68, and HVMCABG) were tightly linked to Alt.
On the other hand, a physiological study has shown that the Al-resistant cultivar of barley secretes citrate from the roots in response to Al (Zhao et al., 2003). The secretion of citrate in barley shows a distinct pattern, which is characterized by a rapid, non-dose-responsive, and temperature-dependent pattern. The secretion is inhibited by anion channel inhibitors, suggesting that citrate secretion is probably mediated through an anion channel on the plasma membrane. A positive correlation between citrate secretion and Al resistance ((root elongation with Al)/(root elongation without Al)) was observed in 21 barley cultivars differing in Al resistance. These results indicate that secretion of citrate is a major mechanism involved in Al resistance in barley. However, it is unknown whether the Al-activated secretion of citrate in Al-resistant barley cultivars is associated with the Al-resistant gene (Alt or Alp) identified genetically as described above. Therefore, the objective of this study was to link the physiology and genetics of Al resistance in barley by mapping the genes responsible for Al resistance and for the Al-activated secretion of citrate.
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Materials and methods |
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Plant materials
Two cultivars of barley (Hordeum vulgare L.), Murasakimochi (CI5899) and Morex, were used, which represent Al-resistant and Al-sensitive cultivars, respectively, according to previous screening results (Zhao et al., 2003). A single Murasakimochi/Morex F1 plant was selfed and 100 randomly chosen F2 plants were used for mapping Al resistance. Those F2 individuals, heterozygous for Al resistance, were selected by the microsatellite markers (Bmac310, Bmag355, and HVM3) linked to the resistance locus which was described in the Results and selfed to generate F3 lines. These F3 lines were planted and checked for heterozygosity by using the SSR markers mentioned above at the Al resistance locus to generate a large F4 mapping population for Al resistance on chromosome 4H. Part of this population was used for mapping citrate secretion in this study.
Measurement of root elongation and citrate secretion
Seeds of Murasakimochi and Morex were soaked in deionized water for 2 h, and then transferred to a Petri dish with moist filter paper and kept in the dark for 24 h at 22–24 °C. Germinated seeds were placed on a net, which was floated on a continuously aerated solution containing 1.0 mM CaCl2 (pH 5.0) in a 1.5 l plastic container. The seedlings were kept in the dark at 22–24 °C for 1 d, and then moved to 25 °C under natural light. The solution was replaced daily. At day 5, the seedlings were exposed to 1.0 mM CaCl2 solution containing 0, 2, 5, and 10 µM AlCl3 at pH 5.0 (Zhao et al., 2003). Root elongation was estimated with 10 replicates by measuring the length of the longest root with a ruler before and after treatment (24 h). Al resistance was expressed as relative root elongation ((root elongation with Al treatment)/(elongation without Al)x100).
For the measurement of citrate secreted from the roots, 12 seedlings (4-d-old) were transplanted to a 1.2 l plastic pot containing aerated 1.0 mM CaCl2 (pH 5.0) overnight, and then exposed to 1.0 mM CaCl2 (pH 5.0) containing 0, 2, 5, and 10 µM AlCl3 for 6 h with three replicates. Root exudates were then collected and passed through a cation exchange resin column (16x14 mm) filled with 5 g of Amberlite IR-120 B resin (H+ form), followed by an anion exchange resin column (16x14 mm) filled with 2 g of AG 1x8 resin (100–200 mesh; formate form). Organic acids retained on the anion exchange resin were eluted with 2 N HCl, and the eluate was concentrated to dryness with a rotary evaporator (40 °C). The residue was dissolved in 1 ml Milli-Q water and subjected to organic acid determination. At the end of the experiment, the number of root apices in each pot was counted and the citrate secretion was expressed on a root (apex) basis.
Citrate in the root exudates was determined according to Delhaize et al. (1993). Briefly, to 1 ml of diluted sample (<50 nmol), 120 µl of 1 M TRIS-HCl buffer (pH 7.8), and 15 µl of 10 mM NADH were added. After incubation at 25 °C for 40 min, 2 µl of enzyme mixture (containing 1.25 units lactate dehydrogenase and 0.5 units malate dehydrogenase) was added and the reaction mixture was incubated for a further 40 min. The absorbance at 340 nm was recorded and, after adding 10 µl citrate lyase (0.5 units), a second recording was taken 15 min later. Standard measurements were performed using the same procedure.
Gene mapping for Al resistance
Al resistance was scored by a ratio of root-to-shoot fresh weight. Ten plants for each parent and 100 plants for the F2 were evaluated. The seeds were germinated for 5 d in Petri dishes at 20 °C. Then, the seedlings were mounted with strips of polyurethane foam on a plastic frame, which covered a 35 l plastic tank containing the nutrient solution, whose composition was described in Raman et al. (2002). Plants were grown in a growth chamber at 20 °C with a 16 h photoperiod and a light intensity of 320 µmol m–2 s–1. After 3 d, the solution was completely changed and 30 µM AlK(SO4)2.12H2O was superimposed for the Al treatment. Throughout the experiment, the nutrient solution was adjusted daily to pH 4.3 with HCl and constantly aerated. After 12 d, the plants were harvested and the fresh weight of roots and shoots were recorded. The ratio of root-to-shoot fresh weight was used as a parameter for ranking Al resistance. Leaves were sampled for DNA extraction.
Leaf DNA was extracted according to the method of Langridge et al. (1997). To identify the molecular markers linked with the Al resistance gene, bulk segregant analysis was carried out by pooling equal amounts of DNA from 10 Al-resistant lines or 10 Al-sensitive lines, based on the result of the Al resistance score described above.
Forty-eight microsatellite markers were selected to examine polymorphism between the Al-resistant and Al-sensitive bulks. These markers were developed by the Scottish Crop Research Institute, and scattered on whole chromosomes. After the identification of linked markers to Al resistance, a Chi-square test was performed among all of the 100 F2 plants to check the goodness of fit in observed numbers of Al-resistant and Al-sensitive individuals into the expected Mendelian segregation ratio.
For further microsatellite analysis, nine markers on chromosome 4H were used including HVM3, HVM13, HVM40, HVM67, HVM68, HVM77, Bmag353, Bmag384, and Bmac310. The details of these makers are described in Liu et al. (1996) and Ramsay et al. (2000). The PCR amplification procedure was described by Costa et al. (2001). The segregation data of Al resistance and microsatellite markers were used for the linkage analyses performed by MAPMAKER/EXP ver. 3.0 (Lander et al., 1987). The Kosambi mapping function was used to calculate map distances (Kosambi, 1944).
Gene mapping for citrate secretion
Two parents and 59 seedlings of F4 (5-d-old) prepared as described above were transplanted to 10 l plastic pots (40 plants per pot) containing continuously aerated nutrient solution. The nutrient solution used was 1/5 strength Hoagland solution (pH 6.0), as reported previously (Ma et al., 1997b). The nutrient solution was replaced once every three days. The plants were grown in a growth chamber with a day/night temperature regime of 25/20 °C for 14/10 h. After a 20 d culture period, plants were exposed to a 1.0 mM CaCl2 solution (pH 5.0) overnight and then each individual plant was exposed to a 1.0 mM CaCl2 solution (pH 5.0) containing 10 µM AlCl3 in a 200 ml pot. After 6 h, the root exudate was collected as described above and roots and shoots were harvested separately. The roots were then dried in an oven at 70 °C for dry weight measurement and the shoots were stored at –80 °C until use. Citrate concentration in the root exudates was assayed as described above.
DNA was extracted according to Langridge et al. (1997). Three microsatelite markers, Bmac310, Bmag353, and HVM3 were used in PCR. The PCR amplification procedure was described by Liu et al. (1996) and Ramsay et al. (2000).
QTL analysis (simple interval mapping) was performed on the data of citrate secretion and polymorphic bands by using the software package MAPMAKER/QTL version 1.1b (Lander and Botstein, 1989). The method detected the linkage between the QTL and a set of markers to scan the whole linkage group. Map distances were calculated using the Kosambi function (Kosambi, 1944).
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Al resistance and citrate secretion
The two cultivars used for preparing the mapping population were characterized in terms of Al resistance and citrate secretion. The root elongation of Murasakimochi was not inhibited at 2 µM Al, while that of Morex was inhibited by 50% (Fig. 1). At higher Al concentrations, root elongation was inhibited more in Morex than in Murasakimochi. When the two cultivars were grown in an acid soil at pH 4.5, the relative root length (root length in acid soil/root length in neutral soil) of Murasakimochi was much higher than that of Morex (data not shown). These results indicate that Murasakimochi is an Al-resistant cultivar, while Morex is an Al-sensitive cultivar, confirming previous screening results (Zhao et al., 2003).
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Citrate secretion from the two cultivars was examined. Murasakimochi secreted a much higher amount of citrate from the roots than Morex in response to Al (Fig. 2). Moreover, when the Al concentrations in the treatment solution increased from 2 µM to 10 µM in Murasakimochi, citrate secretion was only slightly increased. This lack of a distinct dose–response secretion pattern is in agreement with that found in another Al-resistant cultivar, Sigurdkorn (Zhao et al., 2003).
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Gene mapping for Al resistance
Al toxicity is characterized by a rapid inhibition of root elongation, while shoot growth is hardly affected until the second leaf stage, at which point the plant grows independently of the nutrition from the endosperm. However, root elongation or root weight is insufficient to estimate the Al damage of the plant, since growth differences exist among F2 individuals due to parental genetic differences in growth rate. The ratio of root-to-shoot fresh weight was, therefore, used as a parameter for ranking Al resistance in the F2 population. This ratio for Murasakimochi and Morex was 0.39 and 0.17, respectively, and that of the F2 population ranged from 0.11 to 0.60 (Fig. 3). The F2 population could be divided into two at a ratio of 0.24, and Al-resistant lines (>0.24) and Al-sensitive lines (<0.24) segregated at 67:33. According to the Chi-square test, this segregation fits 3:1 (
2=3.41, 0.1<P<0.05). This result indicates that Al resistance is controlled by a single dominant gene.
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Bulk segregant analysis with microsatellite markers showed that the Al resistance gene was localized to a similar position as microsatellite marker Bmag353. Further analysis using microsatellite markers that were known to locate on chromosome 4H revealed that the resistance gene and markers were located in an order of Bmac310 –6.6 cM–Bmag353 (Al resistance) –9.9 cM–HVM3 from the distal side of the short arm on chromosome 4H.
Gene mapping for citrate secretion
Citrate secretion from the roots of Murasakimochi and Morex was 510 and 66 nmol g–1 root dry wt. 6 h–1, respectively, and that of the F4 population ranged from 0 to 881 nmol g–1 root dry wt. 6 h–1 (Fig. 4). Seedlings with a higher citrate secretion (>165 nmol g–1 root dry wt. 6 h–1) and with a lower citrate secretion (<125 nmol g–1 root dry wt. 6 h–1) segregated into 42:17. This segregation ratio also fits 3:1 (2=0.45, 0.50<P<0.75), indicating that the gene for citrate secretion is also controlled by a single dominant gene. However, the continuous segregation of F4 plants may include classification errors between high and low citrate secretion genotypes. Thus, QTL analysis is used to estimate the linkage between a citrate secretion factor and genetic markers.
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To link the gene for Al resistance and for citrate secretion, three microsatellite markers linked with Al resistance (Bmac310, Bmag353, and HVM3) were tested in relation to citrate secretion in 59 F4 seedlings. A segregation pattern of microsatellite marker Bmag353 is shown in Fig. 5. Three bands were observed in heterozygotes. One QTL for Al-activated citrate secretion was detected on chromosome 4H, which was located on the same position as the marker Bmag353 (Fig. 6). This QTL explained 51.3% of the variation for citrate secretion observed among F4 seedlings. This result suggests that the gene for citrate secretion is localized to a position similar to that of the gene for Al resistance (Fig. 6).
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15, F4 seedlings with high citrate secretion, M, molecular marker.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Two cultivars of barley, Murasakimochi and Morex, differed markedly in Al resistance and Al-activated secretion of citrate from the roots (Figs 1, 2). Genetic analysis of the F2 population derived from these two cultivars showed that Al resistance in barley was controlled by a single gene and that this gene was located on chromosome 4H. These results are consistent with previous findings, although the genetic background of the populations and the evaluation method of Al resistance used are quite different (Minella and Sorrells, 1992, 1997; Tang et al., 2000; Raman et al., 2002). For example, Tang et al. (2000) used a population derived from the cross between Dayton (Al-resistant) and Harlan Hybrid (moderately Al-sensitive) and scored Al resistance by the haematoxylin staining method. Raman et al. (2002) used F2 progeny derived from the cross between Yambla (moderately Al-resistant) and WB229 (Al-resistant) and evaluated Al resistance by hydroponic pulse-recovery methods. In the present study, a ratio of root fresh weight to shoot fresh weight was used to score Al resistance. These findings suggest that the inheritance of major Al resistance in barley is simple and the responsible gene is well conserved on chromosome 4H.
Restriction fragment length polymorphism (RFLP) mapping of the Al resistance gene showed that the gene is 2.1 cM proximal to the marker Xbcd117 and 2.1 cM distal to the markers Xwg464 and Xcdo1395 (Tang et al., 2000). Microsatellite analysis showed that the Al resistance gene is closely linked to the marker Bmag353 and Bmac310 (Raman et al., 2002). In the present study, the Al resistance gene was further mapped to a position similar to that of Bmag353 (Fig. 6). As it is still not possible to make a direct comparison between RFLP markers and microsatellite markers, it is not known whether the Al resistance gene mapped by different groups is identical to that on the long arm of chromosome 4H.
On the other hand, the secretion of citrate from the roots has been suggested to be responsible for Al resistance in barley (Zhao et al., 2003). In the present study, QTL analysis for Al-activated secretion of citrate was undertaken using the population derived from the cross between Murasakimochi and Morex. As a result, the gene for citrate secretion was mapped to a position similar to that of the microsatellite marker Bmag353 on chromosome 4H (Fig. 6), where the gene for Al resistance was also located. This result supports the supposition that the secretion of citrate from the roots is the mechanism of Al resistance in barley. As far as is known, this is the first report to integrate the physiology and genetics of Al resistance and link the microsatellite marker to the Al-activated secretion of citrate in barley. In wheat, Al-activated root malate release also co-segregates with the Al resistance locus in wheat (Delhaize et al., 1993) and a gene encoding an Al-activated malate transporter was recently cloned (Sasaki et al., 2004). In an Al-resistant triticale line, the Al-resistant gene, which is mapped to the short arm of chromosome 3R, is associated with organic acid anion secretion (Ma et al., 2000). Recently, Hoekenga et al. (2003) reported that two QTLs for Al resistance co-segregate with an Al-activated release of malate in Arabidopsis roots.
The genetic analysis of Al resistance has also been undertaken in other species of the tribe Triticeae. In wheat, Al resistance in wheat (AltBH) is located on wheat chromosome 4DL (Riede and Anderson, 1996). The dominant loci of Al resistance in rye (Alt1 and Alt3) are located on chromosomes 6R and 4R, respectively (Gallego et al., 1998; Miftahudin et al., 2002). AltBH, Alt3, and Alp genes are suggested to be orthologous loci because of the high level of synteny among chromosomes 4DL, 4RL, and 4HL (Tang et al., 2000; Nguyen et al., 2003), and therefore they may share a common mechanism for Al resistance. In fact, wheat, rye, and barley detoxify Al by secreting organic acid anions from the roots (for a review, see Ma et al., 2001). However, the process leading to the secretion of organic acid anions differs among wheat, barley, and rye. Barley secretes citrate in response to Al and the secretion is characterized by a rapid, temperature-dependent, and non-dose-responsive pattern (Zhao et al., 2003). By contrast, wheat secretes malate and is characterized by a rapid, dose-responsive, but temperature-independent pattern, both malate and citrate secretion in rye is characterized by a delayed, dose-responsive, and temperature-dependent pattern (Li et al., 2000). The amount of organic acid anion secretion also differs following the order rye>wheat>barley, which is consistent with the extent of their Al resistance (Zhao et al., 2003). Hoekenga et al. (2003) indicated that Al-activated secretion of organic acid anions requires the participation of at least three separate cellular processes: (a) perception of toxic Al; (b) synthesis and possibly compartmentation of organic acid anions in the cytosol; and, (c) transport of organic acid anions from the cytosol to the root cell apoplast. Each of the steps could also involve multiple compartments, including different enzymes, transporters, and membrane-associated receptors and other possible signal transduction molecules. Therefore, although the gene for Al-activated secretion of organic acid anions is located on similar chromosomal regions in barley, wheat, and rye, the structure and expression of the gene that regulates the secretion process may be different, resulting in different secretion patterns, different compounds secreted, and different secretion amounts.
The QTL for citrate secretion on chromosome 4H explained more than 50% of the total phenotypic variation in citrate secretion. On the other hand, this result indicated that more than 40% of the variation is unexplained for this trait, which may come from additional physiological factors or experimental errors. The present experiment mainly focused on the identification of function of Al resistance on chromosome 4H and used very targeted plant material segregating only on the region of Al resistance. Additional resistance factors working additively on the present Al resistance will be necessary to raise the level of Al resistance in barley.
The microsatellite marker Bmag353, tightly linked to both the Al-activated secretion of citrate and Al resistance, provides a tool for routine marker-assisted selection. A number of methods of screening for Al resistance have been developed, including replicated field trials and pot assays (Foy, 1996), hydroponic methods (Berzonsky and Kimber, 1986; Ma et al., 1997a), a chlorophyll fluorescence test (Moustakas et al., 1993) and haematoxylin staining (Polle et al., 1978). These methods are labour-intensive, time-consuming, can handle only small populations, and are not able to discriminate heterozygotes (Raman et al., 2002). The marker identified in the present study is PCR-based and is expected to be a rapid, reliable and cost-effective screening system.
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Acknowledgements |
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This study was supported in part by CREST, JST (Japan Science and Technology Cooperation), by a Grant-in-Aid for General Scientific Research (Grant no. 13660067 to JF Ma) from the Ministry of Education, Sports, Culture, Science, and Technology of Japan, and by NSFC (no. 30228023 to JF Ma).
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