Mapping and validation of chromosome regions conferring boron toxicity tolerance in wheat (Triticum aestivum)

Boron is an essential plant micro-nutrient which can be phytotoxic to plants if present in soils in high concentration. Boron toxicity has been recognised as an important problem limiting production in the low rainfall areas of southern Australia, West Asia and North Africa. Genetic variation for boron toxicity tolerance in wheat has been well-characterised. The efficiency of breeding for boron toxicity tolerance could be greatly enhanced by the development of molecular markers associated with QTLs for tolerance in wheat. A population of 161 doubled haploids from a cross between the tolerant cultivar Halberd and the moderately sensitive cultivar Cranbrook was used to identify chromosomal regions involved in boron tolerance. A combined RFLP and AFLP linkage map of the Cranbrook x Halberd population was used to identify chromosomal regions involved in the boron tolerance traits measured. Regions on chromosome 7B and 7D were associated with leaf symptom expression. The region on chromosome 7B was also associated with the control of boron uptake and with a reduction in the effect of boron toxicity on root-growth suppression. RFLP markers at the chromosome 7B and 7D loci were shown to be effective in selecting for improved boron tolerance in an alternative genetic background. Halberd alleles at the chromosome 7B locus were associated with the concentration of boron in whole shoots and grain. The concentration of boron in whole shoots and in grain were both related to grain yield in a field trial conducted on soil containing toxic levels of boron. Implications relating to marker-assisted selection for boron toxicity tolerance in wheat are discussed. Received: 3 September 1999 / Accepted: 12 February 2000     

Assessment of the agronomic value of QTL on chromosomes 2H and 4H linked to tolerance to boron toxicity in barley (Hordeum vulgare L.)

Improved boron (B) tolerance has been an objective of barley breeding programs in regions where B toxicity occurs. Traits associated with B tolerance have been mapped on chromosomes 2H and 4H and it has been proposed that these be used for marker assisted selection for B tolerance. However, there has been little or no improvement in yield using this strategy. This study examined the reasons for the small yield differences among different lines of barley that differ in B tolerance. Experiments used backcross lines derived from crosses between the B-tolerant landrace Sahara 3771 and two adapted recurrent parents, Sloop and VB9104. Lines with different combinations of the Sahara 3771 alleles on chromosomes 2H and 4H were grown over three growing seasons at sites where barley is prone to B toxicity. Grain yields of the backcross lines were similar to or lower than those of the recurrent parents despite showing differences in the expression of B toxicity symptoms and in B concentration in vegetative tissue. There were few significant differences in grain yield among the backcross lines. Variation in dry matter production among the backcross lines in each of the three growing seasons was unrelated to shoot B concentrations while grain yield was correlated with shoot B concentration only among the backcross lines of VB9104 in one season. In this case the yield loss was 4% per 10 mg kg^-1 increase in shoot B concentration. Variation in shoot B concentration and yield across seasons was much greater than that observed among the different barley lines. Reduced B accumulation was associated with higher shoot sodium concentration among the Sloop backcross lines. The results suggest that yield gains from selection based largely on B exclusion and symptoms expression may be small and strongly affected by site and seasonal effects. In the regions where other soil constraints, such as soil salinity and micronutrient deficiencies are also important, reducing B uptake alone may have little effect on yield if these other soil properties are also limiting yields.     

Genes mapping to boron tolerance QTL in barley identified by suppression subtractive hybridization

Boron tolerance is a quantitative trait controlled by multiple genes. Suppression subtractive hybridization was carried out on root cDNA from bulked boron tolerant and intolerant doubled haploid barley lines grown under moderate boron stress to identify genes associated with boron tolerance. One hundred and eleven clones representing known proteins were found to be up‐regulated in the tolerant bulk upon boron stress. Nine clones were genetically mapped to previously reported boron tolerance QTL. These include a clone identical to the boron transporter gene Bot1 and a clone coding for a bromo‐adjacent homology domain‐containing protein, mapping to the 6H boron tolerance locus and co‐segregating with reduced boron intake in a Clipper × Sahara‐3771 mapping population. A third clone mapping to the 2H QTL region encoding an S‐adenosylmethionine decarboxylase precursor was found to provide tolerance to high boron by heterologous expression. Yeast cells expressing Sahara SAMDC were able to grow on 15 mm boron solid media and maintained cellular boron concentrations at 13% lower than control cells expressing empty vector. The data suggest that an antioxidative response mechanism involving polyamines and the ascorbate–glutathione pathway in Sahara barley may provide an advantage in tolerating high soil concentrations of boron.     

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.     

Quantitative trait loci for salinity tolerance in barley (Hordeum vulgare L.)

Salinity stress is a major limitation in barley production. Substantial genetic variation in tolerance occurs among genotypes of barley, so the development of salt-tolerant cultivars is a potentially effective approach for minimizing yield losses. The lack of economically viable methods for screening salinity tolerance in the field remains an obstacle to breeders, and molecular marker-assisted selection is a promising alternative. In this study, salinity tolerance of 172 doubled-haploid lines generated from YYXT (salinity-tolerant) and Franklin (salinity-sensitive) was assessed in glasshouse trials during the vegetative phase. A high-density genetic linkage map was constructed from 76 pairs of simple sequence repeats and 782 Diversity Arrays Technology markers which spanned a total of 1,147 cM. Five significant quantitative trait loci (QTL) for salinity tolerance were identified on chromosomes 1H, 2H, 5H, 6H and 7H, accounting for more than 50% of the phenotypic variation. The tolerant variety, YYXT, contributed the tolerance to four of these QTL and Franklin contributed the tolerance to one QTL on chromosome 1H. Some of these QTL mapped to genomic regions previously associated with salt tolerance in barley and other cereals. Markers associated with the major QTL identified in this study have potential application for marker-assisted selection in breeding for enhanced salt tolerance in barley.     

Boron tolerance in barley is mediated by efflux of boron from the roots

Many plants are known to reduce the toxic effects of high soil boron (B) by reducing uptake of B, but no mechanism for limiting uptake has previously been identified. The B-tolerant cultivar of barley (Hordeum vulgare L.), Sahara, was shown to be able to maintain root B concentrations up to 50% lower than in the B-sensitive cultivar, Schooner. This translated into xylem concentrations that were approximately 64% lower and leaf concentrations 73% lower in the tolerant cultivar. In both cultivars, B accumulation was rapid and reached a steady-state concentration in roots within 3 h. In Schooner, this concentration was similar to the external medium, whereas in Sahara, the root concentration was maintained at a lower concentration. For this to occur, B must be actively extruded from the root in Sahara, and this is presumed to be the basis for B tolerance in barley. The extrusion mechanism was inhibited by sodium azide but not by treatment at low temperature. Several anion channel inhibitors were also effective in limiting extrusion, but it was not clear whether they acted directly or via metabolic inhibition. The ability of Sahara to maintain lower root B concentrations was constitutive and occurred across a wide range of B concentrations. This ability was lost at high pH, and both Schooner and Sahara then had similar root B concentrations. A predictive model that is consistent with the empirical results and explains the tolerance mechanism based on the presence of a borate anion efflux transporter in Sahara is presented.     

Genetic Divergence in Domesticated and Non-Domesticated Gene Regions of Barley Chromosomes

Little is known about the genetic divergence in the chromosomal regions with domesticated and non-domesticated genes. The objective of our study is to examine the effect of natural selection on shaping genetic diversity of chromosome region with domesticated and non-domesticated genes in barley using 110 SSR markers. Comparison of the genetic diversity loss between wild and cultivated barley for each chromosome showed that chromosome 5H had the highest divergence of 35.29%, followed by 3H, 7H, 4H, 2H, 6H. Diversity ratio was calculated as (diversity of wild type – diversity of cultivated type)/diversity of wild type×100%. It was found that diversity ratios of the domesticated regions on 5H, 1H and 7H were higher than those of non-domesticated regions. Diversity ratio of the domesticated region on 2H and 4H is similar to that of non-domesticated region. However, diversity ratio of the domesticated region on 3H is lower than that of non-domesticated region. Averaged diversity among six chromosomes in domesticated region was 33.73% difference between wild and cultivated barley, and was 27.56% difference in the non-domesticated region. The outcome of this study advances our understanding of the evolution of crop chromosomes.     

Identification of boron transporter genes likely to be responsible for tolerance to boron toxicity in wheat and barley

Tolerance to boron (B) toxicity in cereals is known to be associated with reduced tissue accumulation of B. Genes from roots of B-tolerant cultivars of wheat and barley with high similarities to previously reported B efflux transporters from Arabidopsis and rice were cloned. Expression of these genes was strongly correlated with the ability of tolerant genotypes to lower the concentration of B in roots. The gene from barley located to chromosome 4. Backcross lines containing a B tolerance locus on chromosome 4 showed tolerance in proportion to the level of expression of the transporter gene, whereas those lacking the locus were sensitive to B and had very low levels of gene expression. The results are consistent with a widespread mechanism of tolerance to high B based on efflux of B from root cells.     

Uptake of Boron and Silicon by Barley Genotypes with Differing Susceptibilities to Boron Toxicity

In long-term experiments with whole plants, boron uptake bybarley genotypes was linearly related to boron supply over arange of boron concentrations from normal to excessive. Thecomparative susceptibility of genotypes to boron toxicity wasreflected by the relative differences in boron uptake ratesover the entire range of boron supply. Root temperature over the range 5–25 °C markedly affectedplant growth but had no effect on the relative susceptibilityof genotypes to boron toxicity. Concentrations of boron in genotypeswere likewise unaffected by root temperature. Uptake of silicon differed greatly amongst genotypes and alsoreflected the relative susceptibility to boron toxicity andboron uptake. No competitive interaction was observed in theuptake of boron and silicon. The variation in boron accumulation, which governs susceptibilityto boron toxicity amongst barley genotypes, is the result ofdifferences in passive boron uptake via a mechanism which apparentlyalso governs silicon uptake. The nature of this mechanism isdiscussed in relation to the permeability of plant membranesto both boron and silicon. Hordeum vulgare L., barley, boron, toxicity, uptake, silicon, genotypic variation     

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.     

Association mapping of grain hardness,polyphenol oxidase,total phenolics,amylose content,and β-glucan in US barley breeding germplasm

A renewed interest in breeding barley specifically for food end-uses is being driven by increased consumer interest in healthier foods. We conducted association mapping on physicochemical properties of barley that play a role in food quality and processing including grain hardness, polyphenol oxidase activity, total phenolics, amylose content, and β-glucan. We used 3,069 elite two-row and six-row spring barley breeding lines from eight US breeding programs and 2,041 SNP markers for association mapping. Marker–trait associations were identified using a mixed model that incorporated population structure and kinship. We detected two previously identified QTL for grain hardness on chromosome 2H in the telomeric region of 5H along with two novel regions on 4H and 6H. For amylose content, we detected marker–trait associations on 7H from 0.63 to 30 cM. We detected four regions on chromosomes 1H, 2H, 3H, and 4H associated with polyphenol oxidase activity. The chromosome 2H region co-localized with the two previously mapped polyphenol oxidase genes PPO1 and PPO2, and the regions on chromosomes 1H, 3H, and 4H QTL were novel. For total phenolics, we identified three significant regions on 3H, 4H, and 5H. Two regions on 2H and 7H were associated with β-glucan. Both previously identified and novel QTL are segregating in elite US breeding germplasm. Only three of the 24 SNPs that were associated with traits using either the two-row or six-row mapping panel were identified in both panels. Nine SNPs were detected in the individual two-row or six-row panels that were not detected in the analysis using the complete panel and accounting for population structure. The distribution of favorable alleles at these loci that underpin food quality across the breeding programs suggests several strategies to use markers to improve barley for food uses.     

Quantitative Trait Loci for Salinity Tolerance Identified under Drained and Waterlogged Conditions and Their Association with Flowering Time in Barley (Hordeum vulgare. L)

Introduction

Salinity is one of the major abiotic stresses affecting crop production via adverse effects of osmotic stress, specific ion toxicity, and stress-related nutritional disorders. Detrimental effects of salinity are also often exacerbated by low oxygen availability when plants are grown under waterlogged conditions. Developing salinity-tolerant varieties is critical to overcome these problems, and molecular marker assisted selection can make breeding programs more effective.

Methods

In this study, a double haploid (DH) population consisting of 175 lines, derived from a cross between a Chinese barley variety Yangsimai 1 (YSM1) and an Australian malting barley variety Gairdner, was used to construct a high density molecular map which contained more than 8,000 Diversity Arrays Technology (DArT) markers and single nucleotide polymorphism (SNP) markers. Salinity tolerance of parental and DH lines was evaluated under drained (SalinityD) and waterlogged (SalinityW) conditions at two different sowing times.

Results

Three quantitative trait loci (QTL) located on chromosome 1H, single QTL located on chromosomes 1H, 2H, 4H, 5H and 7H, were identified to be responsible for salinity tolerance under different environments. Waterlogging stress, daylight length and temperature showed significant effects on barley salinity tolerance. The QTL for salinity tolerance mapped on chromosomes 4H and 7H, QSlwd.YG.4H, QSlwd.YG.7H and QSlww.YG.7H were only identified in winter trials, while the QTL on chromosome 2H QSlsd.YG.2H and QSlsw.YG.2H were only detected in summer trials. Genes associated with flowering time were found to pose significant effects on the salinity QTL mapped on chromosomes 2H and 5H in summer trials. Given the fact that the QTL for salinity tolerance QSlsd.YG.1H and QSlww.YG.1H-1 reported here have never been considered in the literature, this warrants further investigation and evaluation for suitability to be used in breeding programs.     

DNA methylation pattern in a barley reconstructed karyotype with deleted ribosomal gene cluster of chromosome 6H

A reconstructed barley karyotype (T-35) was utilised to study the influence of chromosomal rearrangements on the DNA methylation pattern at chromosome level. Data obtained were also compared with the distribution of Giemsa N-bands and high gene density regions along the individual chromosomes that have been previously described. In comparison to the control karyotype (T-1586), the DNA methylation pattern was found to vary not only in the reconstructed chromosomes but also in the other chromosomes of the complement. Significant remodelling process of methylation pattern was found also in the residual nucleolus organiser regions (NOR) on chromosome 5H as a consequence of deletion comprising the whole NOR of chromosome 6H in T-35. Moreover, differences between corresponding segments of the homologues with respect to some other chromosome locations were also observed. Repositioning of genomic DNA methylation along the metaphase chromosomes following chromosomal reconstruction in barley seems to be essential to ensure correct chromatin organisation and function.     

Enhanced tolerance to boron toxicity in two-rowed barley by marker-assisted introgression of favourable alleles derived from Sahara 3771

Boron (B) is an essential micronutrient for higher plant, but toxic levels can seriously diminish grain yield in cereal crops by affecting root growth, and thus restricting water extraction from the subsoil. Amelioration of high concentrations in soils is expensive and not always feasible, so breeding for B tolerance is the most viable alternative. This article reports the marker-assisted (MAS) transfer of favourable alleles from an unadapted six-rowed barley (Hordeum vulgare L.) variety, Sahara 3771, into two-rowed lines adapted to southern Australia. During the backcrossing process, the SSR marker, EBmac679, located on chromosome 4H was used to control the target region in foreground selection, but no background selection was applied. Gene introgression was confirmed with 40 BC₆F₁-derived doubled haploid lines segregating for the SSR marker EBmac679. We used a combination of molecular and conventional assays to unequivocally classify the 40 BC₆F₁-derived DH lines as B tolerant or sensitive, and then compared their means for grain yield measured over 2 years and four locations. Results showed modest improvements in grain yield of lines carrying B tolerance genes at some B toxic environments, and negative impact at others. Our results also showed that malting quality profile was not adversely affected through the introgression of the B tolerance allele from Sahara 3771, allowing the newly developed material to be used by breeding programs without risk of a penalty on malt quality.     

Dissection of barley chromosome 5H in common wheat

We dissected barley chromosome 5H added to common wheat by a genetic method or the gametocidal system. Firstly, we induced chromosomal breaks in the offspring of a 5H addition line of common wheat carrying a gametocidal chromosome and cytologically screened for plants with structural chromosomal changes involving 5H, such as deletions and translocations. Secondly, we screened the progeny of such plants to establish common wheat lines carrying structurally changed chromosomes containing single segments of the dissected 5H. Using 23 representative 5H dissection lines, we physically mapped 97 barley EST markers assigned to 5H. The ESTs fell into 20 regions of 5H between the breakpoints of the 23 dissected segments, distributing rather evenly along the chromosome, with significantly higher frequency in the distal region of the long arm. The ESTs, in turn, allowed us to distinguish the breakpoints of dissected 5H segments. We demonstrated by PCR (polymerase chain reaction), as well as by in situ hybridization, that these dissected 5H segments were stably transmitted in the dissection lines. We discuss the usefulness of the 5H dissection lines for physical mapping of DNA markers. These 5H dissection lines are available from National BioResource Projects-Wheat, Japan.     

Comparative sequence analysis of colinear barley and rice bacterial artificial chromosomes

Colinearity of a large region from barley (Hordeum vulgare) chromosome 5H and rice (Oryza sativa) chromosome 3 has been demonstrated by mapping of several common restriction fragment-length polymorphism clones on both regions. One of these clones, WG644, was hybridized to rice and barley bacterial artificial chromosome (BAC) libraries to select homologous clones. One BAC from each species with the largest overlapping segment was selected by fingerprinting and blot hybridization with three additional restriction fragment-length polymorphism clones. The complete barley BAC 635P2 and a 50-kb segment of the rice BAC 36I5 were completely sequenced. A comparison of the rice and barley DNA sequences revealed the presence of four conserved regions, containing four predicted genes. The four genes are in the same orientation in rice, but the second gene is in inverted orientation in barley. The fourth gene is duplicated in tandem in barley but not in rice. Comparison of the homeologous barley and rice sequences assisted the gene identification process and helped determine individual gene structures. General gene structure (exon number, size, and location) was largely conserved between rice and barley and to a lesser extent with homologous genes in Arabidopsis. Colinearity of these four genes is not conserved in Arabidopsis compared with the two grass species. Extensive similarity was not found between the rice and barley sequences other than within the exons of the structural genes, and short stretches of homology in the promoters and 3' untranslated regions. The larger distances between the first three genes in barley compared with rice are explained by the insertion of different transposable retroelements.