An integrated molecular linkage map of diploid wheat based on a <Emphasis Type="Italic">Triticum boeoticum</Emphasis> × <Emphasis Type="Italic">T. monococcum</Emphasis> RIL population

Diploid A genome species of wheat harbour immense variability for biotic stresses and productivity traits, and these could be transferred efficiently to hexaploid wheat through marker assisted selection, provided the target genes are tagged at diploid level first. Here we report an integrated molecular linkage map of A genome diploid wheat based on 93 recombinant inbred lines (RILs) derived from Triticum boeoticum × Triticum monococcum inter sub-specific cross. The parental lines were analysed with 306 simple sequence repeat (SSR) and 194 RFLP markers, including 66 bin mapped ESTs. Out of 306 SSRs tested for polymorphism, 74 (24.2%) did not show amplification (null) in both the parents. Overall, 171 (73.7%) of the 232 remaining SSR and 98 (50.5%) of the 194 RFLP markers were polymorphic. Both A and D genome specific SSR markers showed similar transferability to A genome of diploid wheat species. The 176 polymorphic markers, that were assayed on a set of 93 RILs, yielded 188 polymorphic loci and 177 of these as well as two additional morphological traits mapped on seven linkage groups with a total map length of 1,262 cM, which is longer than most of the available A genome linkage maps in diploid and hexaploid wheat. About 58 loci showed distorted segregation with majority of these mapping on chromosome 2A^m. With a few exceptions, the position and order of the markers was similar to the ones in other maps of the wheat A genome. Chromosome 1A^m of T. monococcum and T. boeoticum showed a small paracentric inversion relative to the A genome of hexaploid wheat. The described linkage map could be useful for gene tagging, marker assisted gene introgression from diploid into hexaploid wheat as well as for map based cloning of genes from diploid A genome species and orthologous genes from hexaploid wheat.     

High molecular weight glutenin subunit in durum wheat (T. durum)

Summary The diversity of high molecular weight (HMW) glutenin subunits of 502 varieties of durum wheat (Triticum durum) from 23 countries was studied using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Twenty-nine types of patterns were observed with 18 mobility bands. A total of 18 alleles were identified by comparing the mobilities of their subunits to those previously found in hexaploid wheat (T. aestivum) and in Triticum turgidum var. dicoccum. Five new alleles were detected: two on the Glu A1 and three on the Glu B1 locus. Comparison of the frequency of alleles in the three species T. aestivum, T. dicoccum and T. durum was investigated. Significant differences exist between each of these species on the basis of the frequency distributions of their three and four common alleles at the Glu A1 and Glu B1 locus, respectively. The Glu B1c allele occuring very frequently in hexaploid wheats was not found in the two tetraploid species. More than 83% of the T. durum analysed were found to have the Glu A1c (null) allele.     

Repetitive, genome-specific probes in wheat (Triticum aestivum L. em Thell) amplified with minisatellite core sequences

The detection and analysis of DNA polymorphisms in crops is an essential component of marker-assisted selection and cultivar identification in plant breeding. We have explored the direct amplification of minisatellite DNA by PCR (DAMD-PCR) as a means for generating DNA probes that are useful for detecting DNA polymorphisms and DNA fingerprinting in wheat. This technique was facilitated by high-stringency PCR with known plant and animal minisatellite core sequences as primers on wheat genomic DNA. The products of DAMD-PCR from Triticum aestivum, T. durum, T. monococcum, T. speltoides and T. tauschii showed a high degree of polymorphism and the various genomes could be identified. Cloning of the DAMD-PCR products and subsequent Southern hybridization frequently revealed polymorphic probes showing a good degree of genome specificity. In addition, polymorphic, single locus, and moderately dispersed PCR products were cloned that may have a potential for DNA fingerprinting. Our experiments were limited primarily to diploid wheats and the results indicated that DAMD-PCR may isolate genome-specific probes from wild diploid wheat species that could be used to monitor genome introgression into hexaploid wheat.This paper reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation for its use by the USDA or the University of Missouri. Contribution from the University of Missouri, the Agricultural Experimental Station and U.S. Department of Agriculture-Agricultural Research Service, Plant Genetics Research Unit, journal series No. 12523     

A chromosome region-specific mapping strategy reveals gene-rich telomeric ends in wheat

A strategy is described for rapid chromosome region-specific mapping in hexaploid wheat (Triticum aestivum L. em. Thell., 2n=6x=42, AABBDD). The method involves allocation of markers to specific chromosome regions by deletion mapping and ordering of probes by high resolution genetic mapping in Triticum tauschii, the D-genome progenitor species. The strategy is demonstrated using 26 chromosome deletion lines for wheat homoeologous group-6. Twenty-five DNA probes from the T. tauschii genetic linkage map and six wheat homoeologous group-6 specific probes were mapped on the deletion lines. Twenty-four of the 25 probes from 6D of T. tauschii also mapped on wheat homoeologous group-6 chromosomes, and their linear order in wheat is the same as in T. tauschii. A consensus physical map of wheat group-6 was constructed because the linear order and the relative position of the probe loci was the same among the three group-6 chromosomes. Comparison of the consensus physical map with the genetic map demonstrated that most of the recombination occurs in the distal ends of the wheat chromosomes. Most of the loci mapped in the distal regions of the chromosomes. The probes were mostly either PstI genomic clones or cDNA clones indicating that the undermethylated single-copy sequences are concentrated in the distal ends of the wheat chromosomes. Fifteen loci are uniformly distributed in the distal 11% of the group-6 chromosomes. Physically, the region spans only 0.58 m, which in wheat translates to about 40 Mb of DNA. The average distance between the markers is, therefore, less than 2.7 Mb and is in the range of PFGE (pulsed-field gel electrophoresis) resolution. Any gene present in the region can be genetically ordered with respect to the markers since the average recombination frequency in the region is very high (>90 cM genetic distance).     

Mechanisms,origin and heredity of Glu-1Ay silencing in wheat evolution and domestication

Key message

Allotetraploidization drives Glu-1Ay silencing in polyploid wheat.

Abstract

The high-molecular-weight glutenin subunit gene, Glu-1Ay, is always silenced in common wheat via elusive mechanisms. To investigate its silencing and heredity during wheat polyploidization and domestication, the Glu-1Ay gene was characterized in 1246 accessions containing diploid and polyploid wheat worldwide. Eight expressed Glu-1Ay alleles (in 71.81% accessions) and five silenced alleles with a premature termination codon (PTC) were identified in Triticum urartu; 4 expressed alleles (in 41.21% accessions), 13 alleles with PTCs and 1 allele with a WIS 2-1A retrotransposon were present in wild tetraploid wheat; and only silenced alleles with PTC or WIS 2-1A were in cultivated tetra- and hexaploid wheat. Both the PTC number and position in T. urartu Glu-1Ay alleles (one in the N-terminal region) differed from its progeny wild tetraploid wheat (1–5 PTCs mainly in the repetitive domain). The WIS 2-1A insertion occurred?~?0.13 million years ago in wild tetraploid wheat, much later than the allotetraploidization event. The Glu-1Ay alleles with PTCs or WIS 2-1A that arose in wild tetraploid wheat were fully succeeded to cultivated tetraploid and hexaploid wheat. In addition, the Glu-1Ay gene in wild einkorn inherited to cultivated einkorn. Our data demonstrated that the silencing of Glu-1Ay in tetraploid and hexaploid wheat was attributed to the new PTCs and WIS 2-1A insertion in wild tetraploid wheat, and most silenced alleles were delivered to the cultivated tetraploid and hexaploid wheat, providing a clear evolutionary history of the Glu-1Ay gene in the wheat polyploidization and domestication processes.

     

Alleles at storage protein loci in Triticum spelta L. accessions and their occurrence in related wheats

The diversity at eight storage protein loci was analyzed in the collection of Triticum spelta accesssions from the National Center for Plant Genetic Resources of Ukraine (most of accessions were European spelts). Seven alleles at the Gli-B1 locus; five alleles at the Gli-A1 and Glu-B1 loci; three alleles at the Glu-B1 locus; and two alleles at the Gli-D1, Gli-B5, Glu-A1, and Glu-D1 loci were identified. Most alleles are found among common wheat cultivars; only five spelt-specific alleles were detected. The high frequency of the GliB1hs* and h alleles encoding the 45-type γ-gliadin among European spelt and durum wheat, as well as the occurrence of these alleles in T. dicoccum (particularly, in emmer accessions from Switzerland and Germany), are evidence in favor of von Büren’s hypothesis that the European spelt arose from the hybridization between tetraploid wheat with the 45-type γ-gliadin and hexaploid wheat. The analysis of genetic distances based on the genotypes at eight storage protein loci allowed differentiating the Asian spelt accession from European spelts.     

Study on the response of diploid,tetraploid and hexaploid species of wheat to the elevated CO2

Study was done to compare the response of Triticum aestivum (hexaploid), Triticum durum (tetraploid) and Triticum monococcum (diploid) wheat species to the elevated CO₂ using Free Air CO₂ Enrichment (FACE) facility. It was demonstrated that the modern cultivar of wheat Triticum aestivum (hexaploid) was largely sink limited. It appeared to have less photosynthesis per unit leaf area than Triticum monococcum (diploid wheat). While leaf size, grain weight and amylase activity increased with the ploidy level from diploid to hexaploid wheat forms, the photosynthetic rate was reduced significantly. These wheat species responded differentially to the elevated CO₂. The larger leaf area and greater seed weight and presence of 38 KDa protein band caused by elevated CO₂ had additive effect in improving the productivity of hexaploid wheat by changing the source sink ratio. Whereas, such a source sink balance was not induced by elevated CO₂ in diploid wheat. The increasing CO₂ may present opportunities to breeders and possibly allow them to select for cultivars responsive to the elevated CO₂ with better sink potential.Key words: Elevated CO₂, FACE technology, Photosynthesis, Seed weight, Source sink ratio, Triticum     

Single-nucleotide polymorphism frequency in a set of selected lines of bread wheat (Triticum aestivum L.).

Information on single-nucleotide polymorphisms (SNPs) in hexaploid bread wheat is still scarce. The goal of this study was to detect SNPs in wheat and examine their frequency. Twenty-six bread wheat lines from different origins worldwide were used. Specific PCR-products were obtained from 21 genes and directly sequenced. SNPs were discovered from the alignment of these sequences. The overall sequence polymorphism observed in this sample appears to be low; 64 single-base polymorphisms were detected in approximately 21.5 kb (i.e., 1 SNP every 335 bp). The level of polymorphism is highly variable among the different genes studied. Fifty percent of the genes studied contained no sequence polymorphism, whereas most SNPs detected were located in only 2 genes. As expected, taking into account a synthetic line created with a wild Triticum tauschii parent increases the level of polymorphism (101 SNPs; 1 SNP every 212 bp). The detected SNPs are available at http://urgi.versailles.inra.fr/GnpSNP">http://urgi.versailles.inra.fr/GnpSNP. Data on linkage disequilibrium (LD) are still preliminary. They showed a significant level of LD in the 2 most polymorphic genes. To conclude, the genome size of hexaploid wheat and its low level of polymorphism complicate SNP discovery in this species.     

Genome-specific primer sets for starch biosynthesis genes in wheat

Common wheat (Triticum aestivum L., 2n=6x=42) is an allohexaploid composed of three closely related genomes, designated A, B, and D. Genetic analysis in wheat is complicated, as most genes are present in triplicated sets located in the same chromosomal regions of homoeologous chromosomes. The goal of this report was to use genomic information gathered from wheat–rice sequence comparison to develop genome-specific primer sets for five genes involved in starch biosynthesis. Intron locations in wheat were inferred through the alignment of wheat cDNA sequences with rice genomic sequence. Exon-anchored primers, which amplify across introns, allowed the sequencing of introns from the three genomes for each gene. Sequence variation within introns among the three wheat genomes provided the basis for genome-specific primer design. For three genes, ADP-glucose pyrophosphorylase (Agp-L), sucrose transporter (SUT), and waxy (Wx), genome-specific primer sets were developed for all three genomes. Genome-specific primers were developed for two of the three genomes for Agp-S and starch synthase I (SsI). Thus, 13 of 15 possible genome-specific primer sets were developed using this strategy. Seven genome-specific primer combinations were used to amplify alleles in hexaploid wheat lines for sequence comparison. Three single nucleotide polymorphisms (SNPs) were identified in a comparison of 5,093 bp among a minimum of ten wheat accessions. Two of these SNPs could be converted into cleaved amplified polymorphism sequence (CAPS) markers. Our results indicated that the design of genome-specific primer sets using intron-based sequence differences has a high probability of success, while the identification of polymorphism among alleles within a genome may be a challenge.     

The inheritance of seed peroxidases of wheat and rye: further data

Summary Further data on the inheritance of seed peroxidases of hexaploid wheat (Triticum aestivum L.) and rye (Secale cereale L.) have been obtained from the genetic analysis of several progenies of both species. Additional data on the inheritance and the chromosomal location and linkage have been obtained for peroxidases of wheat embryo and rye endosperm. The general presence of null alleles in peroxidase loci has been confirmed in both species. In addition to simple monogenic inheritance, epistatic segregations have been observed in both species. These epistatic segregations again suggest the presence of regulatory genes controlling the expression of individual peroxidases in both species and also the existence of several duplicate homoeologous genes in wheat. Known linkage relationships have been confirmed and new ones are indicated. Loci for embryo wheat peroxidases seem to be in chromosomes of the homoeology group 3. The rye endosperm ones should be in chromosome 7R, although it is hypothesized that a duplication of gene EPer1 is located in chromosomes 4R and 7R.     

PCR primed with minisatellite core sequences yields DNA fingerprinting probes in wheat

 Four minisatellite core sequences were used as primers in a polymerase chain reaction (PCR) technique, known as the directed amplification of minisatellite-region DNA (DAMD), to detect polymorphisms in three pairs of hexaploid/tetraploid wheat cultivars. In each pair, the tetraploid cultivar (genomic formula AABB) was extracted from its corresponding hexaploid (genomic formula AABBDD) parent. Reproducible profiles of the amplified products revealed characteristic bands that were present only in the hexaploid wheats but not in their extracted tetraploids. Some polymorphisms were observed among the hexaploid cultivars. Twenty-three DAMD-PCR amplified fragments were isolated and screened as molecular probes on the genomic DNA of wild wheat species, hexaploid wheat and triticale cultivars. Subsequently, 8 of the fragments were cloned and sequenced. The DAMD-PCR clones revealed various degrees of polymorphism among different wild and cultivated wheats. Two clones yielded individual-specific DNA fingerprinting patterns which could be used for species differentiation and cultivar identification. The results demonstrated the use of DAMD-PCR as a tool for the isolation of informative molecular probes for DNA fingerprinting in wheat cultivars and species. Received: 13 May 1996/Accepted: 11 October 1996     

Quality of synthetic hexaploid wheat containing null alleles at Glu-A1 and Glu-B1 loci

Triticum turgidum ssp. dicoccon PI94668 and PI349045 were identified as containing null alleles at Glu-A1 and Glu-B1 loci in previous investigation. Sequencing of the respective HMW-GS genes Ax, Bx, Ay and By in both accessions indicated equal DNA lengths with gene silencing caused by 1 to 4 in-frame stop codon(s) in the open reading frames. Six synthetic hexaploid wheat lines were produced by crossing PI94668 or PI349045 with six Aegilops tauschii by spontaneous chromosome doubling of unreduced gametes. As expected, these amphiploids had three different HMW-GS: Dx 3.1^t?+?Dy11*^t, Dx2.1^t?+?10^t and Dx2^t?+?Dy12^t in Glu-D1 but double nulls in Glu-A1 and Glu-B1. Quality tests showed that most quality parameters in two T. turgidum ssp. dicoccon parents were very low due to the lack of HMW-GSs. However, incorporation of HMW-GS from Ae. tauschii in six synthetic hexaploid wheat lines significantly increased most quality related parameters. The potential values of these wheat lines in improving the quality of wheat are discussed.     

Efficient Agrobacterium transformation of elite wheat germplasm without selection

A previously reported Agrobacterium tumefaciens transformation system that transformed wheat cultivar Fielder at high efficiency was shown to also transform eight out of nine Triticum aestivum (hexaploid wheat) cultivars tested and two Triticum turgidum (durum wheat) cultivars. Transformation efficiencies of these wheat lines ranged from 1.5 to 51 %. Included amongst this germplasm were elite Australian hexaploid wheat cultivars that are currently in commercial cultivation and two of these cultivars, Gladius and Westonia, were transformed at 32 and 45 % efficiency, respectively. Similar high transformation efficiencies were observed for durum wheat cultivars Kronos (51 %) and Stewart (26 %). This highly efficient transformation system was used to generate transgenic plants in the absence of selection and high heritability of unselected transgenes was observed. Selectable marker free transgenic wheat plants were produced at 3 % efficiency. These data demonstrate highly efficient Agrobacterium transformation of diverse wheat germplasm, including elite cultivars, which enables routine production of selectable marker free transgenics.     

About the origin of European spelt (<Emphasis Type="Italic">Triticum spelta</Emphasis> L.): allelic differentiation of the HMW Glutenin B1-1 and A1-2 subunit genes

To investigate the origin of European spelt (Triticum spelta L., genome AABBDD) and its relation to bread wheat (Triticum aestivum L., AABBDD), we analysed an approximately 1-kb sequence, including a part of the promoter and the coding region, of the high-molecular-weight (HMW) glutenin B1-1 and A1-2 subunit genes in 58 accessions of hexa- and tetraploid wheat from different geographical regions. Six Glu-B1-1 and five Glu-A1-2 alleles were identified based on 21 and 19 informative sites, respectively, which suggests a polyphyletic origin of the A- and B-genomes of hexaploid wheat. In both genes, a group of alleles clustered in a distinct, so-called beta subclade. High frequencies of alleles from the Glu-B1-1 and Glu-A1-2 beta subclades differentiated European spelt from Asian spelt and bread wheat. This indicates different origins of European and Asian spelt, and that European spelt does not derive from the hulled progenitors of bread wheat. The conjoint differentiation of alleles of the A- and B-genome in European spelt suggests the introgression of a tetraploid wheat into free-threshing hexaploid wheat as the origin of European spelt.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by J. Dvorak     

Genetic and Molecular Characterization of the VRN2 Loci in Tetraploid Wheat

Winter wheat (Triticum spp.) varieties require long exposures to low temperatures to flower, a process called vernalization. The VRN2 locus includes two completely linked zinc finger-CCT domain genes (ZCCT1 and ZCCT2) that act as flowering repressors down-regulated during vernalization. Deletions or mutations in these two genes result in the elimination of the vernalization requirement in diploid wheat (Triticum monococcum). However, natural allelic variation in these genes has not been described so far in polyploid wheat (tetraploid Triticum turgidum and hexaploid Triticum aestivum). A tetraploid wheat population segregating for both VRN-A2 and VRN-B2 loci facilitated the characterization of different alleles. Comparisons between functional and nonfunctional alleles revealed that both ZCCT1 and ZCCT2 genes are able to confer vernalization requirement and that different ZCCT genes are functional in different genomes. ZCCT1 and ZCCT2 proteins from nonfunctional vrn2 alleles have mutations at arginine amino acids at position 16, 35, or 39 of the CCT domain. These positions are conserved between CCT and HEME ACTIVATOR PROTEIN2 (HAP2) proteins, supporting a model in which the action of CCT domains is mediated by their interactions with HAP2/HAP3/HAP5 complexes. This study also revealed natural variation in gene copy number, including a duplication of the functional ZCCT-B2 gene and deletions or duplications of the complete VRN-B2 locus. Allelic variation at the VRN-B2 locus was associated with a partially dominant effect, which suggests that variation in the number of functional ZCCT genes can be used to expand allelic diversity for heading time in polyploid wheat and, hopefully, improve its adaptation to different environments.     

A study of the storage proteins in the introgression lines of common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L. × <Emphasis Type="Italic">T. timopheevii</Emphasis> Zhuk.) Resistant to brown leaf rust

Storage proteins, prolamins, were studied in ten introgression lines of common wheat bred with involvement of Triticum timopheevii (Tt) Zhuk. and five commercial hexaploid wheat cultivars. The lines are resistant to leaf rust. A comparative analysis of the storage proteins in the Triticum aestivum L. (Ta) introgression lines and the parental forms allowed us to (1) detect the active genes of prolamins on the chromosomes homeologous groups 1 and 6 in the introgression lines of T. aestivum and T. timopheevii; (2) clarify their origin; (3) identify the chromosome attribution of the products; (4) estimate the degree of introgression and postulate the introgression mechanisms; and (5) predict the bread-making quality of these introgression lines.     

Comparative RFLP mapping of the chlorotoluron resistance gene (Su1) in cultivated wheat (Triticum aestivum) and wild wheat (Triticum dicoccoides)

Chlorotoluron is a selective phenylurea herbicide widely used for broad-leaved and annual grass weed control in cereals. Variation in the response to chlorotoluron (CT) was found in both hexaploid bread wheat (Triticum aestivum L.) and wild tetraploid wheat (Triticum dicoccoides KöRN.). Here, we describe the comparative mapping of the CT resistance gene (Su1) on chromosome 6B in bread and wild wheat using RFLP markers. In bread wheat, mapping was based on 58 F₄ single-seed descent (SSD) plants of the cross between a genotype sensitive to chlorotoluron, ‘Chinese Spring’ (CS), and a resistant derivative, the single chromosome substitution line, CS (‘Cappele-Desprez’ 6B) [CS (CAP6B). In T dicoccoides, mapping was based on 37 F₂ plants obtained from the cross between the CT-susceptible accession B-7 and the resistant accession B-35. Nine RFLP probes spanning the centromere were chosen for mapping. In bread wheat Su1 was found to be linked to α-Amy-1 (9.84 cM) and Xpsr371 (5.2 cM), both on the long arm of 6B, and Nor2 (2.74 cM) on the short arm. In wild wheat the most probable linkage map was Nor2-Xpsr312-Su1-Pgk2, and the genetic distances between the genes were 24.8cM, 5.3cM, and 6.8cM, respectively. These results along with other published map data indicate that the linear order of the genes is similar to that found in T. aestivum. The results of this study also show that the Su1 gene for differential response to chlorotoluron has evolved prior to the domestication of cultivated wheat and not in response to the development and use of chemicals.     

Allelic variation of high molecular weight glutenin subunits of bread wheat in Hebei province of China

In common wheat (Triticum aestivum L.), allelic variations of Glu-1 loci have important influences on grain end-use quality. The allelic variations in high molecular weight glutenin subunits (HMW-GSs) were identified in 151 hexaploid wheat varieties representing a historical trend in the cultivars introduced or released in Hebei province of China from the years 1970s to 2010s. Thirteen distinct alleles were detected for Glu-1. At Glu-A1, Glu-B1 and Glu-D1, we found that the most frequent alleles were the 1 (43.0%), 7+8 (64.9%), 2+12 (74.8%) alleles, respectively, in wheat varieties. Twenty two different HMW-GS compositions were observed in wheat. Twenty-five (16.6%) genotypes possessed the combination of subunits 1, 7+8, 2+12, 25 (16.6%) genotypes had subunit composition of 2*, 7+8, 2+12; 20 (13.2%) genotypes had subunit composition of null, 7+8, 2+12. The frequency of other subunit composition was less than 10%. The Glu-1 quality score greater than or equal to 9 accounted for 20.6% of the wheat varieties. The percentage of superior subunits (1 or 2* subunit at Glu-A1 locus; 7+8, 14+15 or 17+18 at Glu-B1 locus; 5+10 or 5+12 at Glu-D1 locus) was an upward trend over the last 40 years. The more different superior alleles correlated with good bread-making quality should be introduced for their usage in wheat improvement efforts.     

Genetic diversity for morphological traits and seed storage proteins in Spanish rivet wheat

The objective of the current work was to analyse the variability of high and low molecular mass (HMM and LMM) glutenin subunits, along with some morphological characteristics in sixty Spanish accessions of rivet wheat (Triticum turgidum L. ssp. turgidum). The lines were grouped in sixteen botanical varieties and five additional types, according the morphological criteria. Up to 13 allelic variants (four alleles for the Glu-A1 locus and nine alleles for the Glu-B1 locus) and 34 B-LMMGs patterns were found in the evaluated lines. The current data indicated a clear reduction of morphological variability, along with an asymmetric distribution of the alleles and patterns for seed storage proteins. This polymorphism could be useful for enlarging the genetic background of modern durum wheat.