Molecular mapping of a gene responsible for Al-activated secretion of citrate in barley

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.     

Development of gene-specific markers for acid soil/aluminium tolerance in barley (Hordeum vulgare L.)

Acid soil/aluminium toxicity is one of the major constraints on barley production around the world. Genetic improvement is the best solution and molecular-marker-assisted selection has proved to be an efficient tool for developing barley cultivars with acid soil/aluminium tolerance. In this study, barley variety Svanhals—introduced from CYMMIT (International Maize and Wheat Improvement Center)—was identified as acid soil/aluminium tolerant and the tolerance was mapped to chromosome 4H in 119 doubled haploid (DH) lines from a cross of Hamelin/Svanhals. The HvMATE gene, encoding an aluminium-activated citrate transporter, was selected as a candidate gene and gene-specific molecular markers were developed to detect acid soil/aluminium tolerance based on the polymerase chain reaction. Sequence analysis of the HvMATE gene identified a 21-bp indel (insertion–deletion) between the tolerant and sensitive cultivars. The new marker was further mapped to the QTL (quantitative trait loci) region on chromosome 4H for acid soil tolerance and accounted for 66.9 % of phenotypic variation in the DH population. Furthermore, the polymorphism was confirmed in other tolerant varieties which have been widely used as a source of acid soil tolerance in Australian barley breeding programs. The new gene-specific molecular marker provides an effective and simple molecular tool for selecting the acid soil tolerance gene from multiple tolerance sources.     

A new aluminum tolerance gene located on rye chromosome arm 7RS

Rye has one of the most efficient groups of genes for aluminum tolerance (Alt) among cultivated species of Triticeae. This tolerance is controlled by, at least, three independent and dominant loci (Alt1, Alt2, and Alt3) located on chromosome arms 6RS, 3RS, and 4RL, respectively. The segregation of Alt genes and several random amplified polymorphic DNA (RAPD), Secale cereale inter-microsatellite (SCIM), and Secale cereale microsatellite (SCM) markers in three F(2) between a tolerant cultivar (Ailés) and a non-tolerant inbred line (Riodeva) were studied. The segregation ratio obtained for aluminum tolerance in the three F(2) populations analyzed was 3:1 (tolerant:non-tolerant), indicating that tolerance is controlled by one dominant locus. SCIM811(1376) was linked to an Alt gene in the three F(2) populations studied, and three different SCIMs and one RAPD (SCIM811(1376), SCIM812(626), SCIM812(1138), and OPQ4(725)) were linked to the Alt gene in two F(2) populations. This result indicated that the same Alt gene was segregating in the three crosses. SCIM819(1434) and OPQ4(578) linked to the tolerance gene in one F(2) population were located using wheat-rye ditelosomic addition lines on the 7RS chromosome arm. The Alt locus is mapped between SCIM819(1434) and the OPQ4(578) markers. Two microsatellite loci (SCM-40 and SCM-86), previously located on chromosome 7R, were also linked to the Alt gene. Therefore, the Alt gene segregating in these F(2) populations is new and probably could be orthologous to the Alt genes located on wheat chromosome arm 4DL, on barley chromosome arm 4HL, on rye chromosome arm 4RL, and rice chromosome 3. This new Alt gene located on rye chromosome arm 7RS was named Alt4. A map of rye chromosome 7R with the Alt4 gene, 16 SCIM and RAPD, markers and two SCM markers was obtained.     

Fine-mapping of the BaMMV, BaYMV-1 and BaYMV-2 resistance of barley (Hordeum vulgare) accession PI1963

Barley yellow mosaic disease caused by the bymoviruses barley mild mosaic virus (BaMMV) and barley yellow mosaic virus (BaYMV) is one of the economically most important diseases of winter barley in Europe. In European barley breeding programmes, resistance is currently due to only two genes—rym4, which is effective against viruses BaMMV and BaYMV-1, and rym5, which is effective against BaYMV-2. Diversification of resistance is therefore an important task. Because the accession PI1963 confers immunity against all European strains of barley yellow mosaic disease and is not allelic to rym5, we have attempted to develop closely linked markers in order to facilitate the efficient introgression of this resistance into adapted germplasm. By means of restriction fragment length polymorphism analysis, we located a gene locus for resistance to BaMMV, BaYMV-1 and BaYMV-2 of PI1963 on chromosome 4HL using a mapping population (W757) comprising 57 doubled haploid (DH) lines. Subsequent tests for allelism indicated that the BaMMV resistance gene in PI1963 is allelic to rym11. Two DH populations, IPK1 and IPK2, comprising 191 and 161 DH lines, respectively, were derived from the initial mapping population W757 and used for further analysis. As random amplified polymorphic DNA development did not facilitate the identification of more closely linked markers, simple sequence repeat (SSR) analyses were conducted. For population IPK1, the closest SSRs detected were Bmac181 and Bmag353, which flank the gene at 2.1 cM and 2.7 cM, respectively. For the IPK2 population, the SSR markers HVM3 and Bmag353 are located proximally at 2.5 cM and distally at 8.2 cM, respectively. In order to develop markers more tightly linked to rym11, a targeted amplified fragment length polymorphism (AFLP) marker identification approach was adopted using bulks comprising lines carrying recombination events proximal and distal to the target interval. Using this approach we identified six AFLP markers closely linked to rym11, with the two markers, E56M32 and E49M33, co-segregating with rym11 in both populations. The SSRs and AFLPs identified in this study represent useful tools for marker-assisted selection.     

Search for microsatellite markers associated with water-stress tolerance in wheat through bulked segregant analysis

We used bulked segregant analysis (BSA) to identify microsatellite markers associated with water-stress tolerance in wheat. Two DNA pools (tolerant and sensitive) were established from the selected F₂ individuals of crosses between water-stress-tolerant and -sensitive wheat parental genotypes on the basis of the paraquat (PQ) tolerance, leaf size, and relative water content. All three traits were previously shown to be associated with water-stress tolerance on segregating F₂ progeny of the wheat crosses used in this study. Microsatellite analysis was then performed on the established DNA pools, using 35 primer pairs that included all of the chromosome group 5 (5A, 5B, 5D) markers, to detect microsatellite fragments that were present, absent, or both in the DNA pools and their parental lines. We identified one microsatellite fragment that was present in tolerant parent wheat and the tolerant bulk but absent in the sensitive parent wheat and sensitive bulk. We then followed the segregation of this marker in the tolerant F₂ individuals. Use of this marker may significantly enhance the success of selection for PQ- and water-stress-tolerant genotypes in wheat breeding programs.     

AFLP markers tightly linked to the aluminum-tolerance gene Alt3 in rye (Secale cereale L.)

Rye (Secale cereale L.) is considered to be the most aluminum (Al)-tolerant species among the Triticeae. It has been suggested that aluminum tolerance in rye is controlled by three major genes (Alt genes) located on rye chromosome arms 3RL, 4RL, and 6RS, respectively. Screening of an F₆ rye recombinant inbred line (RIL) population derived from the cross between an Al-tolerant rye (M39A-1–6) and an Al-sensitive rye (M77A-1) showed that a single gene controls aluminum tolerance in the population analyzed. In order to identify molecular markers tightly linked to the gene, we used a combination of amplified fragment length polymorphism (AFLP) and bulked segregant analysis techniques to evaluate the F₆ rye RIL population. We analyzed approximately 22,500 selectively amplified DNA fragments using 204 primer combinations and identified three AFLP markers tightly linked to the Alt gene. Two of these markers flanked the Alt locus at distance of 0.4 and 0.7 cM. Chromosomal localization using cloned AFLP and a restriction fragment length polymorphism (RFLP) marker indicated that the gene was on the long arm of rye chromosome 4R. The RFLP marker (BCD1230) co-segregated with the Alt gene. Since the gene is on chromosome 4R, the gene was designated as Alt3. These markers are being used as a starting point in the construction of a high resolution map of the Alt3 region in rye. Received: 29 March 2000 / Accepted: 9 July 2001     

High-resolution mapping of the <Emphasis Type="Italic">Alp</Emphasis> locus and identification of a candidate gene <Emphasis Type="Italic">HvMATE</Emphasis> controlling aluminium tolerance in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Aluminium (Al) tolerance in barley is conditioned by the Alp locus on the long arm of chromosome 4H, which is associated with Al-activated release of citrate from roots. We developed a high-resolution map of the Alp locus using 132 doubled haploid (DH) lines from a cross between Dayton (Al-tolerant) and Zhepi 2 (Al-sensitive) and 2,070 F₂ individuals from a cross between Dayton and Gairdner (Al-sensitive). The Al-activated efflux of citrate from the root apices of Al-tolerant Dayton was 10-fold greater than from the Al-sensitive parents Zhepi 2 and Gairdner. A suite of markers (ABG715, Bmag353, GBM1071, GWM165, HvMATE and HvGABP) exhibited complete linkage with the Alp locus in the DH population accounting 72% of the variation for Al tolerance evaluated as relative root elongation. These markers were used to map this genomic region in the Dayton/Gairdner population in more detail. Flanking markers HvGABP and ABG715 delineated the Alp locus to a 0.2 cM interval. Since the HvMATE marker was not polymorphic in the Dayton/Gairdner population we instead investigated the expression of the HvMATE gene. Relative expression of the HvMATE gene was 30-fold greater in Dayton than Gardiner. Furthermore, HvMATE expression in the F_2:3 families tested, including all the informative recombinant lines identified between HvGABP and ABG715 was significantly correlated with Al tolerance and Al-activated citrate efflux. These results identify HvMATE, a gene encoding a multidrug and toxic compound extrusion protein, as a candidate controlling Al tolerance in barley.     

Development of PCR-based codominant markers flanking the Alt3 gene in rye.

Aluminum (Al) toxicity is considered to be a major problem for crop growth and production on acid soils. The ability of crops to overcome Al toxicity varies among crop species and cultivars. Rye (Secale cereale L.) is the most Al-tolerant species among the Triticeae. Our previous study showed that Al tolerance in a rye F6 recombinant inbred line (RIL) population was controlled by a single gene designated as the aluminum tolerance (Alt3) gene on chromosome 4RL. Based on the DNA sequence of a rice (Oryza sativa L.) BAC clone suspected to be syntenic to the Alt3 gene region, we developed two PCR-based codominant markers flanking the gene. These two markers, a sequence-tagged site (STS) marker and a cleaved amplified polymorphic sequence (CAPS) marker, each flanked the Alt3 gene at an approximate distance of 0.4 cM and can be used to facilitate high-resolution mapping of the gene. The markers might also be used for marker-assisted selection in rye or wheat (Triticum aestivum L.) breeding programs to obtain Al-tolerant lines and (or) cultivars.     

Candidate gene identification of an aluminum-activated organic acid transporter gene at the Alt4 locus for aluminum tolerance in rye (Secale cereale L.)

Among cereal crops, rye is one of the most tolerant species to aluminum. A candidate gene approach was used to determine the likely molecular identity of an Al tolerance locus (Alt4). Using PCR primers designed from a wheat aluminum tolerance gene encoding an aluminum-activated malate transporter (TaALMT1), a rye gene (ScALMT1) was amplified, cloned and sequenced. Subsequently, the ScALMT1 gene of rye was found to be located on 7RS by PCR amplification using the wheat–rye addition lines. SNP polymorphisms for this gene were detected among the parents of three F₂ populations that segregate for the Alt4 locus. A map of the rye chromosome 7R, including the Alt4 locus ScALMT1 and several molecular markers, was constructed showing a complete co-segregation between Alt4 and ScALMT1. Furthermore, expression experiments were carried out to clarify the function of this candidate gene. Briefly, the ScALMT1 gene was found to be primarily expressed in the root apex and upregulated when aluminum was present in the medium. Five-fold differences in the expression were found between the Al tolerant and the Al non-tolerant genotypes. Additionally, much higher expression was detected in the rye genotypes than the moderately tolerant “Chinese Spring” wheat cultivar. These results suggest that the Alt4 locus encodes an aluminum-activated organic acid transporter gene that could be utilized to increase Al tolerance in Al sensitive plant species. Finally, TaALMT1 homologous sequences were identified in different grasses and in the dicotyledonous plant Phaseolus vulgaris. Our data support the hypothesis of the existence of a common mechanism of Al tolerance encoded by a gene located in the homoeologous group four of cereals. G. Fontecha and J. Silva-Navas contributed equally to this work.     

运用近等基因系（NIL）、AFLP、RFLP和SCAR标记对玉米S组育性恢复基因（Rf3）的研究

以1对近等基因系（NIL）及其回交群体（BC     

Molecular markers linked to the aluminium tolerance gene Alt1 in rye (Secale cereale L.)

 Rye has one of the most efficient group of genes for aluminium (Al) tolerance among cultivated species of Triticeae. This tolerance is controlled by at least two independent and dominant loci (Alt1 and Alt3) located on chromosomes 6RS and 4R. We used two pooled DNA samples, one of Al-tolerant individuals and another of Al-sensitive plants from one F₂ that segregated for the Alt1 locus. We also used two pooled DNA samples, one with genotypes 11 and another with genotypes 22 for the Lap1 locus (leucin aminopeptidase) from another F₂ progeny that segregated for this locus, located on the 6RS chromosome arm. We identified several RAPD markers associated with the pooled Al-tolerant plants and also with one of the bulks for the Lap1 locus. The RAPD fragments linked to Alt1 and Lap1 genes were transformed into SCAR markers to confirm their chromosomal location and linkage data. Two SCARs (ScR01 ₆₀₀ and ScB15 ₇₉₀₀ ) were closely linked to the Alt1 locus, ScR01 ₆₀₀ located 2.1 cM from Alt1 and ScB15 ₇₉₀ located 5.5 cM from Alt1, on the 6RS chromosome arm. These SCAR markers can aid in the transfer of Al tolerance genes into Al-sensitive germplasms. Received: 9 December 1997 / Accepted: 12 May 1998     

Detection and mapping of SSRs in rye ESTs from aluminium-stressed roots

Aluminium toxicity is a major problem for crop production on acid soils. Rye (Secale cereale L.) has one of the most efficient group of genes for aluminium tolerance, at least, four independent and dominant loci, Alt1, Alt2, Alt3 and Alt4, located on chromosome arms 6RS, 3RS, 4RL and 7RS, have been described. The increasing availability of expressed sequence tags in rye and related cereals provides a valuable resource of non-anonymous DNA molecular markers. In order to obtain simple sequence repeat (SSR) markers related with Al tolerance more than 1,199 public accessible rye cDNA sequences from Al-stressed roots were exploited as a resource for SSR markers development. From a total of 21 S. cereale microsatellite (SCM) loci analysed, 12 were located on chromosomes 1R, 2R, 3R, 4R and 5R, using wheat–rye addition lines or mapped using a F₂ population segregating for Al tolerance. Seven SCM loci were included in a rye map with other SCIM and RAPD markers. Moreover, 14 SCM loci could be associated to proteins with known or unknown function. The possible implications of these sequences in aluminium tolerance mechanisms are discussed.     

A rapid hydroponic screening for aluminium tolerance in barley

Selection and breeding of crops for aluminium (Al) tolerance is a useful approach to increase production on acid soils. This requires a rapid and reliable system to discriminate between Al-tolerant and Al-sensitive genotypes. A hydroponic system was developed to screen for Al tolerance in barley (t Hordeum vulgare L.) to overcome several problems encountered in previous screening methods. Four levels of Al (5, 10, 20, and 40 t M) in 1 mt M CaCl₂ solution at pH 4.5 were used to rank lines for Al-tolerance. Each line was cultured in a different compartment to eliminate chemical and pH interactions among lines. To avoid changes in Al tolerance due to other factors such as the calcium (Ca) concentration of the solution, Al-tolerant (Atlas 66) and Al-sensitive (Scout 66) cultivars of wheat (t Triticum aestivum L.) were used as reference cultivars. Five ranks of Al tolerance from highly tolerant to highly sensitive were established by comparison with each reference. Eriochrome cyanine R staining was used for the rapid evaluation of Al tolerance. This screening system allowed classification of about 50 barley lines into five different Al tolerance groups within one week. Using this system, screening of ca. 600 barley lines from various regions of the world was conducted. Most lines were sensitive to Al, but ninety lines showed intermediate Al-tolerance. Thirty nine lines were highly sensitive to Al in solution.     

Comparative mapping of a major aluminum tolerance gene in sorghum and other species in the poaceae

In several crop species within the Triticeae tribe of the grass family Poaceae, single major aluminum (Al) tolerance genes have been identified that effectively mitigate Al toxicity, a major abiotic constraint to crop production on acidic soils. However, the trait is quantitatively inherited in species within other tribes, and the possible ancestral relationships between major Al tolerance genes and QTL in the grasses remain unresolved. To help establish these relationships, we conducted a molecular genetic analysis of Al tolerance in sorghum and integrated our findings with those from previous studies performed in crop species belonging to different grass tribes. A single locus, AltSB, was found to control Al tolerance in two highly Al tolerant sorghum cultivars. Significant macrosynteny between sorghum and the Triticeae was observed for molecular markers closely linked to putatively orthologous Al tolerance loci present in the group 4 chromosomes of wheat, barley, and rye. However, AltSB was not located within the homeologous region of sorghum but rather mapped near the end of sorghum chromosome 3. Thus, AltSB not only is the first major Al tolerance gene mapped in a grass species that does not belong to the Triticeae, but also appears to be different from the major Al tolerance locus in the Triticeae. Intertribe map comparisons suggest that a major Al tolerance QTL on rice chromosome 1 is likely to be orthologous to AltSB, whereas another rice QTL on chromosome 3 is likely to correspond to the Triticeae group 4 Al tolerance locus. Therefore, this study demonstrates a clear evolutionary link between genes and QTL encoding the same trait in distantly related species within a single plant family.     

Water stress mediated root trait dynamics and identification of microsatellite markers associated with root traits in rice ( Oryza sativa L.)

To identify microsatellite markers associated with root traits for drought tolerance in rice (Oryza sativa L.) a study was conducted at Department of Plant Physiology, College of Agriculture, Trivandrum, Kerala Agricultural University. A set of thirty-five rice genotypes were exposed to water stress and evaluated for physio-morphological components as indices of water stress tolerance. Observations were made on leaf rolling score and root traits, especially the root length, root dry weight, root volume and root shoot ratio at booting stage. As of the data obtained, ten tolerant and ten susceptible varieties were selected for bulk line analysis to identify the DNA markers linked with target gene conferring drought tolerance. Out of 150 SSR primers screened, RM474 showed polymorphism between the tolerant and susceptible bulks. Individual genotypes of the bulks also showed the same product size of the respective tolerant and susceptible bulks.     

Genetic and physical characterization of chromosome 4DL in wheat.

The long arm of chromosome 4D in wheat (Triticum aestivum L.) has been shown in previous studies to harbor genes of agronomic importance. A major dominant gene conferring Aluminum (Al) tolerance (Alt2 in 'Chinese Spring' and AltBH in 'BH 1146'), and the Knal locus controlling the K+/Na+ discrimination in saline environments have been mapped to this chromosome arm. However, accurate information on the genetic and physical location of markers related to any of these genes is not available and would be useful for map-based cloning and marker-assisted plant breeding. In the present study, using a population of 91 recombinant inbred lines segregating for Al tolerance, we provide a more extensive genetic linkage map of the chromosome arm 4DL based on RFLP, SSR, and AFLP markers, delimiting the AltBH gene to a 5.9-cM interval between markers Xgdm125 and Xpsr914. In addition, utilizing a set of wheat deletion lines for chromosome arm 4DL, the AltBH gene was physically mapped to the distal region of the chromosome, between deletion breakpoints 0.70 and 0.86, where the kilobase/centimorgan ratio is assumed to be low, making the map-based cloning of the gene a more realistic goal. The polymorphism rates in chromosome arm 4DL for the different types of markers used were extremely low, as confirmed by the physical mapping of AFLPs. Finally, analysis of 1 Mb of contiguous sequence of Arabidopsis chromosome 5 flanking the gene homologous to the BCD1230 clone (a cosegregating marker in our population coding for a ribulose-5-phosphate-3-epimerase gene), revealed a previously identified region of stress-related and disease-resistance genes. This could explain the collinearity observed in comparative mapping studies among different species and the low level of polymorphism detected in the chromosome arm 4DL in hexaploid wheat.     

Molecular mapping of the shrunken endosperm genes seg8 and sex1 in barley (Hordeum vulgare L.).

A number of mutations affecting seed development in barley (Hordeum vulgare L.) have been known for many years; however, to date, no research has been reported that elucidates the molecular structure of the causal genes. As a first step, we initiated the linkage mapping of the two shrunken endosperm genes seg8 and sex1 using microsatellite markers. The recessive gene seg8 was mapped in the centromeric region of chromosome 7H to a 4.6 cM interval flanked by markers GBM1516 and Bmag341. The recessive sex1 gene showed xenia effects and was located in the centromeric region of barley chromosome 6H, which is in accordance to the previously reported chromosomal location in the classical linkage map. It was flanked by markers GBM5012 and GBM1063 in a 4.2 cM interval. EST-derived microsatellite markers were used to establish the syntenic relationships to the genomic rice sequences. Two orthologous sites on rice chromosome 2 flanking a 4.1 Mb sequence had homology to the respective barley markers in the sex1 region. For the markers in the seg8 region orthologous sites on rice chromosome 6 were detected.     

Physical analysis of the complex rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.) <Emphasis Type="Italic">Alt4</Emphasis> aluminium (aluminum) tolerance locus using a whole-genome BAC library of rye cv. Blanco

Rye is a diploid crop species with many outstanding qualities, and is important as a source of new traits for wheat and triticale improvement. Rye is highly tolerant of aluminum (Al) toxicity, and possesses a complex structure at the Alt4 Al tolerance locus not found at the corresponding locus in wheat. Here we describe a BAC library of rye cv. Blanco, representing a valuable resource for rye molecular genetic studies, and assess the library’s suitability for investigating Al tolerance genes. The library provides 6 × genome coverage of the 8.1 Gb rye genome, has an average insert size of 131 kb, and contains only ~2% of empty or organelle-derived clones. Genetic analysis attributed the Al tolerance of Blanco to the Alt4 locus on the short arm of chromosome 7R, and revealed the presence of multiple allelic variants (haplotypes) of the Alt4 locus in the BAC library. BAC clones containing ALMT1 gene clusters from several Alt4 haplotypes were identified, and will provide useful starting points for exploring the basis for the structural variability and functional specialization of ALMT1 genes at this locus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identifying aluminum tolerance in rice with a molecular marker

Improving rice aluminum (Al) tolerance in acid soils is highly significant to rice production. So far, no molecular marker which can efficiently identify rice Al tolerance has been developed. In this study, Art1, a primer specific for the ART1 gene was designed to study whether some natural variations exist in ART1 between Al tolerant and sensitive varieties. The results showed that two Al tolerant varieties had identical nucleotides and amino acids, while three Al sensitive varieties had the same nucleotides and amino acids differing from the two Al tolerant varieties. The same results were discovered within 150 rice varieties of Ting’s core collection. Art1 could be used to judge rice Al tolerance.