Molecular characterization and mapping of ALMT1, the aluminium-tolerance gene of bread wheat (Triticum aestivum L.).

The major aluminum (Al) tolerance gene in wheat ALMT1 confers. An Al-activated efflux of malate from root apices. We determined the genomic structure of the ALMT1 gene and found it consists of 6 exons interrupted by 5 introns. Sequencing a range of wheat genotypes identified 3 alleles for ALMT1, 1 of which was identical to the ALMT1 gene from an Aegilops tauschii accession. The ALMT1 gene was mapped to chromosome 4DL using 'Chinese Spring' deletion lines, and loss of ALMT1 coincided with the loss of both Al tolerance and Al-activated malate efflux. Aluminium tolerance in each of 5 different doubled-haploid populations was found to be conditioned by a single major gene. When ALMT1 was polymorphic between the parental lines, QTL and linkage analyses indicated that ALMT1 mapped to chromosome 4DL and cosegregated with Al tolerance. In 2 populations examined, Al tolerance also segregated with a greater capacity for Al-activated malate efflux. Aluminium tolerance was not associated with a particular coding allele for ALMT1, but was significantly correlated with the relative level of ALMT1 expression. These findings suggest that the Al tolerance in a diverse range of wheat genotypes is primarily conditioned by ALMT1.     

Analysis of TaALMT1 traces the transmission of aluminum resistance in cultivated common wheat (Triticum aestivum L.)

Allele diversities of four markers specific to intron three, exon four and promoter regions of the aluminum (Al) resistance gene of wheat (Triticum aestivum L.) TaALMT1 were compared in 179 common wheat cultivars used in international wheat breeding programs. In wheat cultivars released during the last 93 years, six different promoter types were identified on the basis of allele size. A previous study showed that Al resistance was not associated with a particular coding allele for TaALMT1 but was correlated with blocks of repeated sequence upstream of the coding sequence. We verified the linkage between these promoter alleles and Al resistance in three doubled haploid and one intercross populations segregating for Al resistance. Molecular and pedigree analysis suggest that Al resistance in modern wheat germplasm is derived from several independent sources. Analysis of a population of 278 landraces and subspecies of wheat showed that most of the promoter alleles associated with Al resistance pre-existed in Europe, the Middle East and Asia prior to dispersal of cultivated germplasm around the world. Furthermore, several new promoter alleles were identified among the landraces surveyed. The TaALMT1 promoter alleles found within the spelt wheats were consistent with the hypothesis that these spelts arose on several independent occasions from hybridisations between non-free-threshing tetraploid wheats and Al-resistant hexaploid bread wheats. The strong correlation between Al resistance and Al-stimulated malate efflux from the root apices of 49 diverse wheat genotypes examined was consistent with the previous finding that Al resistance in wheat is conditioned primarily by malate efflux. These results demonstrate that the markers based on intron, exon and promoter regions of TaALMT1 can trace the inheritance of the Al resistance locus within wheat pedigrees and track Al resistance in breeding programmes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Sequence and tissue-specific expression of a putative peroxidase gene from wheat (Triticum aestivum L.)

We have used a cDNA clone encoding a pathogen-induced putative wheat peroxidase to screen a genomic libary of wheat (Triticum aestivum L. cv. Cheyenne) and isolated one positive clone, lambda POX1. Sequence analysis revealed that this clone contains a gene encoding a putative peroxidase with a calculated pI of 8.1 which exhibits 58% and 83% sequence identity to the amino acid sequence of the turnip (Brassica rapa) peroxidase and a pathogen-induced putative wheat peroxidase, respectively. The two introns in the wheat gene are at the same positions as introns in the peroxidase genes of tomato and horseradish. Results of S1-mapping experiments suggest that this gene is neither pathogen-nor wound-induced in leaves but is constitutively expressed in roots.     

Inheritance of cell size in wheat (Triticum aestivum L.) and its relationship to the vernalization loci

Reduced cell size is an important adaptive feature in plant response to environmental stresses. The objectives of the present study were to determine the inheritance and location of genes controlling cell size and to establish the relationship between cell size, low-temperature (LT) tolerance, and growth habit as determined by the Vrn loci in wheat. Guard cell length was measured in F₁, F₂, andF₂-derived F₃ populations from parents ranging widely in cell size and in the Chinese Spring/ Cheyenne (CS/CNN) chromosome substitution series. The cell size of F₁ hybrids was similar to the parental midpoint and the F₂ frequency distribution was symmetrical about the mean indicating that cell size was determined by additive gene action with little or no dominance. It appears that there are several genes involved since none of the F₂ progeny had a cell size as large or as small as the parental mean range. The cell size of the homozygous spring and winter lines from F₂-derived F₃ populations fell into two distinct groups that were related to plant growth habit. Large cell size was associated with the spring-habit alleles (Vrn-A1) and small cell size was associated with the winter-habit alleles (vrn-A1) on chromosome 5A. Analyses of the CS/CNN chromosome substitution series showed that CNN chromosomes 5A and 5B both reduced cell size without changing the growth habit, indicating that growth habit per se does not determine cell size. The group-5 chromosomes therefore appear to carry homoeologous alleles with major effects on cell size in wheat. This places cell-size control and many other low-temperature (LT) tolerance associated characters in close proximity to the vrn region of the group-5 chromosomes. Received: 17 August 2000 / Accepted: 20 November 2000     

Mapping of the loose smut resistance gene Ut6 in wheat (Triticum aestivum L.)

Loose smut of wheat (Triticum aestivum L.) caused by Ustilago tritici (Pers.) Rostr. can cause considerable yield losses in the absence of appropriate management practices. The use of wheat varieties with loose smut resistance is an efficient and effective control technique. However, the development of commercial wheat lines with resistance to loose smut is time- and labour-consuming. DNA markers linked to loose smut resistance gene(s) would assist the development of loose smut resistant genotypes. The genetics of loose smut resistance was studied in an F5‐derived recombinant inbred line (RIL) population of 94 lines from the cross BW278/AC Foremost. The line AC Foremost is resistant and line BW278 is susceptible to U. tritici race T10. Phenotypic assessment revealed that a single gene, designated Ut6, segregated for resistance to race T10 in the RIL population. A modified bulked segregant analysis identified a microsatellite marker linked to Ut6. A linkage map was developed consisting of linked microsatellite loci and the resistance gene. The loose smut resistance gene Ut6 mapped to the long arm of chromosome 5B. Five microsatellite markers mapped within 6.7 cM of Ut6. The microsatellite markers gpw5029 and barc232 flanked Ut6 at distances of 1.3 and 2.8 cM on the distal and proximal sides, respectively. A diverse set of wheat lines was haplotyped for Ut6 using the linked microsatellite markers gpw5029 and barc232. The haplotype analysis suggested that the microsatellite markers associated with Ut6 will be useful for marker-assisted selection of loose smut resistant wheat lines.     

The wheat (Triticum aestivum L.) leaf proteome

The wheat leaf proteome was mapped and partially characterized to function as a comparative template for future wheat research. In total, 404 proteins were visualized, and 277 of these were selected for analysis based on reproducibility and relative quantity. Using a combination of protein and expressed sequence tag database searching, 142 proteins were putatively identified with an identification success rate of 51%. The identified proteins were grouped according to their functional annotations with the majority (40%) being involved in energy production, primary, or secondary metabolism. Only 8% of the protein identifications lacked ascertainable functional annotation. The 51% ratio of successful identification and the 8% unclear functional annotation rate are major improvements over most previous plant proteomic studies. This clearly indicates the advancement of the plant protein and nucleic acid sequence and annotation data available in the databases, and shows the enhanced feasibility of future wheat leaf proteome research.     

Silicon absorption by wheat (Triticum aestivum L.)

Although silicon (Si) is a quantitatively major inorganic constituent of higher plants the element is not considered generally essential for them. Therefore it is not included in the formulation of any of the solution cultures widely used in plant physiological research. One consequence of this state of affairs is that the absorption and transport of Si have not been investigated nearly as much as those of the elements accorded 'essential' status. In this paper we report experiments showing that Si is rapidly absorbed by wheat (Triticum aestivum L.) plants from solution cultures initially containing Si at 0.5 mM, a concentration realistic in terms of the concentrations of the element in soil solutions. Nearly mature plants (headed out) 'preloaded' with Si absorbed it at virtually the same rate as did plants grown previously in solutions to which Si had not been added. The rate of Si absorption increased by more than an order of magnitude between the 2-leaf and the 7-8 leaf stage, with little change thereafter. This revised version was published online in June 2006 with corrections to the Cover Date.     

Five genes induced by aluminum in wheat (Triticum aestivum L.) roots.

Five different cDNAs (termed wali1 to wali5 for Wheat Aluminum Induced) whose expression was induced by Al stress have been isolated from the root tips of Al-treated wheat (Triticum aestivum L.) plants. Four of these genes were induced 24 to 96 h after Al treatment, and their expression is reduced when the Al is removed. Each of these four genes was induced by inhibitory levels of Al in two wheat cultivars--Warigal, an Al-sensitive cultivar, and Waalt, an Al-tolerant cultivar. The fifth gene (wali2) showed a complex bimodal pattern of induction and was induced by Al only in the sensitive cultivar. Comparison of the nucleotide sequences of these clones to those in the sequence data bases showed that wali4 is homologous to phenylalanine ammonia-lyase and wali1 is homologous to a group of plant proteins that are cysteine-rich and have homology to metallothioneins. wali2 encodes a novel protein with a repeating motif of cysteine amino acids. The remaining two wali clones (wali3 and wali5) encode related, cysteine-rich proteins that show no significant homology to any known sequences.     

Repetitive Indel Markers within the ALMT1 Gene Conditioning Aluminium Tolerance in Wheat (Triticum aestivum L.)

ALMT1 gene encoding a membrane protein that facilitates an aluminium stimulated malate efflux has been characterised and mapped in wheat (Triticum aestivum L.). Here, we have identified molecular markers targeting insertion/deletion (indel) and SSR repeats within intron 3 region of the ALMT1 gene. Both the markers: ALMT1-SSR3a and ALMT1-SSR3b based on repetitive indels, exhibited complete cosegregation with Al tolerance, malate efflux, and a CAPS marker discriminating ALMT1-1 and ALMT1-2 alleles, in a doubled haploid population derived from Diamondbird (Al-tolerant)/Janz (Al-sensitive). A parental screen of 20 diverse wheat genotypes with repetitive indel markers indicated that six allele variants exist at the ALMT1SSR3 locus. Sequence analysis confirmed that these variations were due to indels, copy number of SSR repeats, and base substitution within SSR repeats. The higher level of variation in intron 3 suggests that this genomic region has been constrained by indels, SSR and single nucleotide polymorphisms. Results have proven that repetitive indel markers cosegregating with the Al tolerance locus will be useful for marker assisted selection and population and evolution studies.     

Microsatellite mapping of a Triticum urartu Tum. derived powdery mildew resistance gene transferred to common wheat (Triticum aestivum L.)

A powdery mildew resistance gene from Triticum urartu Tum. accession UR206 was successfully transferred into hexaploid wheat (Triticum aestivum L.) through crossing and backcrossing. The F₁ plants, which had 28 chromosomes and an average of 5.32 bivalents and 17.36 univalents in meiotic pollen mother cells (PMC), were obtained through embryos rescued owing to shriveling of endosperm in hybrid seed of cross Chinese Spring (CS) × UR206. Hybrid seeds were produced through backcrossing F₁ with common wheat parents. The derivative lines had normal chromosome numbers and powdery mildew resistance similar to the donor UR206, indicating that the powdery mildew resistance gene originating from T. urartu accession UR206 was successfully transferred and expressed in a hexaploid wheat background. Genetic analysis indicated that a single dominant gene controlled the powdery mildew resistance at the seedling stage. To map and tag the powdery mildew resistance gene, 143 F₂ individuals derived from a cross UR206 × UR203 were used to construct a linkage map. The resistant gene was mapped on the chromosome 7AL based on the mapped microsatellite makers. The map spanned 52.1 cM and the order of these microsatellite loci agreed well with the established microsatellite map of chromosome arm 7AL. The resistance gene was flanked by the microsatellite loci Xwmc273 and Xpsp3003, with the genetic distances of 2.2 cM and 3.8 cM, respectively. On the basis of the origin and chromosomal location of the gene, it was temporarily designated PmU.     

Nuclear and cytoplasmic gene control of resistance to loose smut (Ustilago tritici (Pers.) Rostr.) in wheat (Triticum aestivum L.)

Summary Using disomic chromosome substitution lines based on the susceptible wheat cultivar Chinese Spring, loose smut resistance of wheat cultivars Hope and Thatcher was shown to be conferred in each case by a single dominant major gene carried on chromosome 7 A (Hope) or 7 B (Thatcher). Partial resistance was determined by genes on an additional eight Hope or seven Thatcher chromosomes, and similarities were evident between the partial resistance genotypes ofHope and Thatcher. Chinese Spring exhibited a mean infection value of approximately 50%, indicating a significant level of partial resistance, which was found to be due, in part, to genes on the homoeologous chromosome arms 1 As, 1 Es and 1 Ds, and to cytoplasmic genes. Substitution of the Chinese Spring nucleus into the cytoplasm of Aegilops squarrosa, Ae. variabilis or Ae. mutica resulted in increased susceptibility to Ustilago tritici. Several alloplasmic lines of the resistant wheat cultivars Selkirk and Chris exhibited race-specific susceptibility to U. tritici.     

Genetic and physical characterization of theLR1 leaf rust resistance locus in wheat (Triticum aestivum L.)

The objective of this study was to characterize the leaf rust resistance locusLr1 in wheat. Restriction fragment length polymorphism (RELP) analysis was performed on the resistant lineLr1/6^*Thatcher and the susceptible varieties Thatcher and Frisal, as well as on the segregating F₂ populations. Seventeen out of 37 RFLP probes mapping to group 5 chromosomes showed polymorphism betweenLr1/6^*Thatcher and Frisal, whereas 11 probes were polymorphic between the near-isogenic lines (NILs)Lr1/6^*Thatcher and Thatcher. Three of these probes were linked to the resistance gene in the segregating F₂ populations. One probe (pTAG621) showed very tight linkage toLr1 and mapped to a single-copy region on chromosome 5D. The map location of pTAG621 at the end of the long arm of chromosome 5D was confirmed by the absence of the band in the nulli-tetrasomic line N5DT5B of Chinese Spring and a set of deletion lines of Chinese Spring lacking the distal part of 5DL. Twenty-seven breeding lines containing theLr1 resistance gene in different genetic backgrounds showed the same band asLr1/6^*Thatcher when hybridized with pTAG621. The RFLP marker was converted to a sequence-tagged-site marker using polymerase chain reaction (PCR) amplification. Sequencing of the specific fragment amplified from both NILs revealed point mutations as well as small insertion/deletion events. These were used to design primers that allowed amplification of a specific product only from the resistant lineLr1/6^*Thatcher. This STS, specific for theLr1 resistance gene, will allow efficient selection for the disease resistance gene in wheat breeding programmes. In addition, the identification of a D-genome-specific probe tightly linked toLr1 should ultimately provide the basis for positional cloning of the gene.     

Molecular identification of powdery mildew resistance genes in common wheat (Triticum aestivum L.)

RFLP markers for the wheat powdery mildew resistance genes Pm1 and Pm2 were tagged by means of near-isogenic lines. The probe Whs178 is located 3 cM from the Pm1 gene. For the powdery mildew resistance gene Pm2, two markers were identified. The linkage between the Pm2 resistance locus and one of these two probes was estimated to be 3 cM with a F₂ population. Both markers can be used to detect the presence of the corresponding resistance gene in commercial cultivars. Bulked segregant analysis was applied to identify linkage disequillibrium between the resistance gene Pm18 and the abovementioned marker, which was linked to this locus at a distance of 4 cM. Furthermore, the RAPD marker OPH-11₁₉₀₀ (5-CTTCCGCAGT-3) was selected with pools created from a population segregating for the resistance of Trigo BR 34. The RAPD marker was mapped about 13 cM from this resistance locus.     

Accelerated production of transgenic wheat (Triticum aestivum L.) plants

We have developed a method for the accelerated production of fertile transgenic wheat (Triticum aestivum L.) that yields rooted plants ready for transfer to soil in 8–9 weeks (56–66 days) after the initiation of cultures. This was made possible by improvements in the procedures used for culture, bombardment, and selection. Cultured immature embryos were given a 4–6 h pre-and 16 h post-bombardment osmotic treatment. The most consistent and satisfactory results were obtained with 30 g of gold particles/bombardment. No clear correlation was found between the frequencies of transient expression and stable transformation. The highest rates of regeneration and transformation were obtained when callus formation after bombardment was limited to two weeks in the dark, with or without selection, followed by selection during regeneration under light. Selection with bialaphos, and not phosphinothricin, yielded more vigorously growing transformed plantlets. The elongation of dark green plantlets in the presence of 4–5 mg/l bialaphos was found to be reliable for identifying transformed plants. Eighty independent transgenic wheat lines were produced in this study. Under optimum conditions, 32 transformed wheat plants were obtained from 2100 immature embryos in 56–66 days, making it possible to obtain R3 homozygous plants in less than a year.     

Fine mapping Fhb1, a major gene controlling fusarium head blight resistance in bread wheat (Triticum aestivum L.)

A major fusarium head blight (FHB) resistance gene Fhb1 (syn. Qfhs.ndsu-3BS) was fine mapped on the distal segment of chromosome 3BS of spring wheat (Triticum aestivum L.) as a Mendelian factor. FHB resistant parents, Sumai 3 and Nyubai, were used as sources of this gene. Two mapping populations were developed to facilitate segregation of Qfhs.ndsu-3BS in either a fixed resistant (Sumai 3*5/Thatcher) (S/T) or fixed susceptible (HC374/3*98B69-L47) (HC/98) genetic background (HC374 = Wuhan1/Nyubai) for Type II resistance. Type II resistance (disease spread within the spike) was phenotyped in the greenhouse using single floret injections with a mixture of macro-conidia of three virulent strains of Fusarium graminearum. Due to the limited heterogeneity in the genetic background of the crosses and based on the spread of infection, fixed recombinants in the interval between molecular markers XGWM533 and XGWM493 on 3BS could be assigned to discrete “resistant” and “susceptible” classes. The phenotypic distribution was bimodal with progeny clearly resembling either the resistant or susceptible parent. Marker order for the two maps was identical with the exception of marker STS-3BS 142, which was not polymorphic in the HC/98 population. The major gene Fhb1 was successfully fine mapped on chromosome 3BS in the same location in the two populations within a 1.27-cM interval (S/T) and a 6.05-cM interval (HC/98). Fine mapping of Fhb1 in wheat provides tightly linked markers that can reduce linkage drag associated with marker-assisted selection of Fhb1 and assist in the isolation, sequencing and functional identification of the underlying resistance gene.     

Agrobacterium-mediated In-planta transformation of bread wheat (Triticum aestivum L.)

Journal of Plant Biochemistry and Biotechnology - Agrobacterium-mediated in-planta transformation method allows efficient plant transformation without tissue culture. In the present study, a tissue...     

Suspension and protoplast culture of hexaploid wheat (Triticum aestivum L.)

Suspension cultures have been initiated from embryogenic callus of hexaploid wheat (Triticum aestivum L.). Most commonly, these suspensions are composed of callus-like clusters (up to 2 mm in diameter). Two rapidly-growing lines (MBE6 and C82d) have been obtained, which consist of smaller aggregates of cytoplasmic cells, and these have been maintained for more than 4 years. These lines show very limited morphogenetic capacity and only a single plantlet has been regenerated, from line MBE6, after 9 months in culture. Protoplasts isolated from line MBE6 are unable to divide, but protoplasts from line C82d consistently undergo sustained divisions to form callus or secondary cell suspensions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium     

Assembling complex genotypes to resist Fusarium in wheat (Triticum aestivum L.)

Fusarium head blight of wheat is a major deterrent to wheat production world-wide. The genetics of FHB resistance in wheat are becoming clear and there is a good understanding of the genome location of FHB resistance QTL from different sources such as Sumai3, Wuhan, Nyubai and Frontana. All the components needed for assembling complex genotypes through large-scale molecular breeding experiments are now available. This experiment used high throughput microsatellite genotyping and half-seed analysis to process four independent crosses through a molecular breeding strategy to introduce multiple pest resistance genes into Canadian wheat. This included two backcrosses and selection for a total of six FHB resistance QTL, orange blossom wheat midge resistance (Sm1) and leaf rust resistance (Lr21). In addition, the fixation of the elite genetic background was monitored with 45–76 markers to accelerate restoration of the genetic background at each backcross. The strategy resulted in 87% fixation of the elite genetic background on average at the BC₂F₁ generation and successfully introduced all of the chromosome segments containing FHB, Sm1 and Lr21 resistance genes. The molecular breeding strategy was completed in 25 months, at an equal pace to conventional crossing and selection of spring wheat.