Genetic mapping of two powdery mildew resistance genes in einkorn (Triticum monococcum L.) accessions

Powdery mildew is a severe foliar disease for wheat and could cause great yield loss in epidemic years. To explore new powdery mildew resistance genes, two einkorn accessions including TA2033 and M80, both resistant to this disease, were studied for the inheritance of resistance. Each accession possessed a single but different dominant resistance gene that was designated as Mlm2033 and Mlm80, respectively. Marker mapping indicated that they are both linked to Xgwm344 on the long arm of chromosome 7A. To establish their genetic relationship with Pm1 on 7AL, five RFLP markers previously reported to co-segregate with Pm1a were converted to STS markers. Three of them detected polymorphism between the mapping parents and were mapped close to Mlm2033 or Mlm80 or both. Xmag2185, the locus determined by the STS marker derived from PSR680, one of the RFLP markers, was placed less than 2 cM away from them. The allelism test indicated that Mlm2033 and Mlm80 are likely allelic to each other. In addition, through comparative and EST mapping, more markers linked to these two genes were identified. The high density mapping of Mlm2033 and Mlm80 will contribute to map-based cloning of the Pm1 locus. The markers for both genes will also facilitate their transfer to wheat.     

High molecular weight glutenin subunit variation in wild and cultivated einkorn wheats (Triticum spp.,Poaceae)

Variation in high molecular weight (HMW) glutenin subunit composition among wild and cultivated einkorn wheats (2n = 2x = 14, AA) was investigated using one- (SDS-PAGE and urea/SDS-PAGE) and two-dimensional (IEF × SDS-PAGE) electrophoretic analyses. The material comprised 150 accessions ofTriticum urartu, 160 accessions ofT. boeoticum, 24 accessions ofT. boeoticum subsp.thaoudar and 74 accessions of primitive domesticatedT. monococcum from many different germplasm collections. The biochemical characteristics of HMW-glutenin subunits ofT. boeoticum andT. monococcum were highly similar to one another but distinctly different from those ofT. urartu. All the species analysed were characterised by large intraspecific variation and only three HMW-glutenin subunit patterns were identical betweenT. boeoticum andT. monococcum. Consistent with the distinct nature ofT. urartu, all its HMW-glutenin patterns were different from those found inT. boeoticum andT. monococcum. The differences detected between these species might reflect their reproductive isolation and are consistent with recent nomenclatural and biosystematic treatments that recogniseT. urartu as separate species fromT. boeoticum andT. monococcum. The presence of three distinct glutenin components in some accessions of the species studied seems to be evidence for the existence of at least three active genes controlling the synthesis of the HMW-glutenin subunits in the A genome of wild and primitive domesticated diploid wheats. Results indicate also that HMW-glutenin subunits could represent useful markers for the evaluation of genetic variability present in different wild diploid wheat collections and subsequently for their conservation and future utilisation.     

Wx gene in diploid wheat: molecular characterization of five novel alleles from einkorn (Triticum monococcum L. ssp. monococcum) and T. urartu

The Wx gene encodes the granule-bound starch synthase I or waxy protein, which is the sole enzyme responsible for amylose synthesis in wheat seeds. Triticum urartu and einkorn (T. monococcum L. ssp. monococcum), which are related to the A genome of bread wheat, could be important sources of variation for this gene. This study evaluated the Wx gene variability in 52 accessions of these species and compared their nucleotide sequences with the Wx-A1a allele of bread wheat. The level of polymorphism found was high, although not distributed equally between the two species. Five different alleles were found in T. urartu, of which four were novel (Wx-A ^u 1b, -A ^u 1c, -A ^u 1d and -A ^u 1e). All einkorn accessions had the same allele, which was also novel and was named Wx-A ^m 1a. A comparison between the proteins deduced from the novel alleles and the Wx-A1a protein showed that there were up to 33 amino acid changes in both the transit peptide and the mature protein. These results showed that these species, especially T. urartu, are a potential source of novel waxy variants.     

Isolation and characterization of five novel high molecular weight subunit of glutenin genes from Triticum timopheevi and Aegilops cylindrica

Analysis by SDS-PAGE of total protein fractions from single seeds of Aegilops cylindrica (genomes C and D) and Triticum timopheevi (genomes A and G) showed the presence of three bands corresponding to high molecular weight subunits of glutenin (HMW subunits) in the former and two major bands and a minor band corresponding to HMW subunits in the latter. Three Ae. cylindrica and two T. timopheevi HMW subunit gene sequences, each comprising the entire coding region, were amplified by polymerase chain reaction (PCR) and their complete nucleotide sequences determined. A combination of N-terminal amino acid sequencing of the proteins identified by SDS-PAGE and alignments of the derived amino acid sequences of the proteins encoded by the PCR products identified the Ae. cylindrica HMW subunits as 1Cx, 1Cy and 1Dy, and the T. timopheevi HMW subunits as 1Gx, 1Ax and 1Ay. It was not clear whether or not a 1Gy HMW subunit was present in T. timopheevi. The PCR products from Ae. cyclindrica were derived from 1Cy and 1Dy genes and a silent 1Dx gene containing an in-frame internal stop codon, while those from T. timopheevi were derived from 1Ax and 1Ay genes. The 1Cx, 1Gx and 1Gy sequences were not amplified successfully. The proteins encoded by the five novel genes had similar structures to previously characterized HMW subunits of bread wheat (Triticum aestivum). Differences and similarities in sequence and structure, and in the distribution of cysteine residues (relevant to the ability of HMW subunits to form high M_r polymers) distinguished the HMW subunits of x- and y-type and of each genome rather than those of the different species. There was no evidence of a change in HMW subunit expression or structure resulting from selective breeding of bread wheat. The novel 1Ax, 1Ay, 1Cy and 1Dy HMW subunits were expressed in Escherichia coli, and the expressed proteins were shown to have very similar mobilities to the endogenous HMW subunits on SDS-PAGE. The truncated 1Dx gene from Ae. cylindrica failed to express in E. coli, and no HMW subunit-related protein of the size predicted for the truncated 1Dx subunit could be identified by immunodetection in seed extracts.     

In vitro mature embryo culture protocol of einkorn (Triticum monococcum ssp. monococcum) and bread (Triticum aestivum L.) wheat under boron stress

Mature embryos of einkorn (Triticum monococcum ssp. monococcum) and bread (Triticum aestivum L.) wheat were used for callus induction on media containing four different doses (0, 1, 2 and 4 mg L^?1) of 2,4-D and dicamba supplemented with five different boron concentrations (0, 6.2, 12.4, 24.8, and 37.2 mg L^?1). The obtained callus was transferred to culture media with three (0, 0.5, and 2 mg L^?1) different BAP doses with five boron concentrations for further regeneration. The maximum callus weight in einkorn wheat was in culture media with 1 mg L^?1 dicamba and 6.2 mg L^?1 (3.71?±?0.13 g). Bread wheat had the maximum callus weight on culture media with 4 mg L^?1 dicamba and 12.4 mg L^?1 (3.46?±?0.40 g). The highest plantlet numbers were in only 2 mg L^?1 BAP (2.92?±?0.88) for einkorn wheat and 0.5 mg L^?1 BAP supplemented with 6.2 mg L^?1 boron (3.71?±?1.12) for bread wheat. This indirect regeneration protocol using mature embryos of einkorn and bread wheat under boron stresses expected to be useful for future wheat breeding studies.

     

Molecular characterization and genomic organization of low molecular weight glutenin subunit genes at the Glu-3 loci in hexaploid wheat (Triticum aestivum L.)

In this study, we report on the molecular characterization and genomic organization of the low molecular weight glutenin subunit (LMW-GS) gene family in hexaploid wheat (Triticum aestivum L.). Eighty-two positive BAC clones were identified to contain LMW-GS genes from the hexaploid wheat ‘Glenlea’ BAC library via filter hybridization and PCR validation. Twelve unique LMW glutenin genes and seven pseudogenes were isolated from these positive BAC clones by primer-template mismatch PCR and subsequent primer walking using hemi-nested touchdown PCR. These genes were sequenced and each consisted of a single-open reading frame (ORF) and untranslated 5′ and 3′ flanking regions. All 12 LMW glutenin subunits contained eight cysteine residues. The LMW-m-type subunits are the most abundant in hexaploid wheat. Of the 12 LMW-GS, 1, 2 and 9 are i-type, s-type and m-type, respectively. The phylogenetic analysis suggested that the LMW-i type gene showed greater differences to LMW-s and LMW-m-type genes, which, in turn, were more closely related to one another. On the basis of their N-terminal sequences, they were classified into nine groups. Fingerprinting of the 82 BAC clones indicated 30 BAC clones assembled into eight contigs, while the remaining clones were singletons. BAC end sequencing of the 82 clones revealed that long terminal repeat (LTR) retrotransposons were abundant in the Glu-3 regions. The average physical distance between two adjacent LMW-GS genes was estimated to be 81 kb. Most of LMW-GS genes are located in the d-genome, suggesting that the Glu-D3 locus is much larger than the Glu-B3 locus and Glu-A3 locus. Alignments of sequences indicated that the same type (starting with the same N-terminal sequence) LMW-GS genes were highly conserved in the homologous genomes between hexaploid wheat and its donors such as durum wheat and T. tauschii. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

栽培一粒小麦3AA30中抗白粉病基因的鉴定及分子标记定位

栽培一粒小麦是普通小麦的近缘种,遗传多样性丰富,蕴含丰富的抗病基因,是小麦抗病性改良的重要资源。本文对栽培一粒小麦抗白粉病材料3AA30的抗白粉病基因进行了遗传分析和分子标记定位。结果表明,3AA30中含有一个隐性抗白粉病基因,暂命名为ml3AA30,找到了5个与该基因连锁的SSR分子标记Xgwm6、Xcfd39、Xcfa2185、Xcfa2141、Xcfa2155及2个STS标记Xmag2170、Xmag1491,并构建了ml3AA30的遗传连锁图,将该基因定位在小麦5A染色体长臂上。本研究为小麦抗病育种提供了新的抗源材料。     

Molecular cloning and comparative analysis of a y-type inactive HMW glutenin subunit gene from cultivated emmer wheat (Triticum dicoccum L.)

Cultivated emmer (Triticum dicoccum, 2n = 4x = 28, AABB) is closely related to bread wheat and possesses extensive allelic variations in high molecular weight glutenin subunit (HMW-GS) composition. These alleles may be an important genetic resource for wheat quality improvement. To isolate and clone HMW-GS genes from cultivated emmer, two pairs of allele-specific (AS) PCR primers were designed to amplify the coding sequence of y-type HMW-GS genes and their upstream sequences, respectively. The results showed that single bands of strong amplification were obtained through AS-PCR of genomic DNA from emmer. After cloning and sequencing the complete sequence of coding and 5'-flanking regions of a y-type subunit gene at Glu-A1 locus was obtained. Nucleotide and deduced amino acid sequences analysis showed that this gene possessed a similar structure as the previously reported Ay gene from common wheat, and is hence designated as Ay1d. The distinct feature of the Ay1d gene is that its coding region contains four stop codons and its upstream region has a 85-bp deletion in the same position of the Ay gene, which are probably responsible for the silencing of y-type subunit genes at Glu-A1 locus. Phylogenetic analysis of HMW glutenin subunit genes from different Triticum species and genomes were also carried out.     

Molecular cloning and characterization of four novel LMW glutenin subunit genes from Aegilops longissima, Triticum dicoccoides and T. zhukovskyi

This paper reports cloning and characterisation of four novel low-molecular-weight glutenin subunit (LMW-GS) genes (designated as TzLMW-m2, TzLMW-m1, TdLMW-m1 and AlLMW-m2) from the genomic DNA of Triticum dicoccoides, T. zhukovskyi and Aegilops longissima. The coding regions of TzLMW-m2, TzLMW-m1, TdLMW-m1 and AlLMW-m2 were 1056 bp, 903 bp, 1056 bp and 1050 bp in length, encoding 350, 300, 350 and 348 amino acid residues, respectively. The deduced amino acid sequences showed that the four novel genes were classified as LMW-m types and the comparison results indicated that the four genes had a more similar structure and a higher level of homology with the LMW-m genes than the LMW-s and -i types genes. However, the first cysteine residue's positions of TzLMW-m2, TdLMW-m1 and AlLMW-m2 were different from the others. Moreover, AlLMW-m2, TdLMW-m1 and TzLMW-m2 all possessed a longer repetitive domain, which was considered to be associated with good quality of wheat. The secondary structure prediction revealed that the content of beta-strand in AlLMW-m2 and TdLMW-m1 exceeded the positive control, suggesting that AlLMW-m2 and TdLMW-m1 should be considered as candidate genes that may have positive effect on dough quality. In order to investigate the evolutionary relationship of the novel genes with the other LMW-GSs, a phylogenetic tree was constructed. The results lead to a speculation that AlLMW-m2, TdLMW-m1 and TzLMW-m2 may be the middle types during the evolution of LMW-m and LMW-s.     

Characterization of low-molecular-weight glutenin subunit Glu-B3 genes and development of STS markers in common wheat (Triticum aestivum L.)

Low-molecular-weight glutenin subunit (LMW-GS) Glu-B3 has a significant influence on the processing quality of the end-use products of common wheat. To characterize the LMW-GS genes at the Glu-B3 locus, gene-specific PCR primers were designed to amplify eight near-isogenic lines and Cheyenne with different Glu-B3 alleles (a, b, c, d, e, f, g, h and i) defined by protein electrophoretic mobility. The complete coding regions of four Glu-B3 genes with complete coding sequence were obtained and designated as GluB3-1, GluB3-2, GluB3-3 and GluB3-4. Ten allele-specific PCR markers designed from the SNPs present in the sequenced variants discriminated the Glu-B3 proteins of electrophoretic mobility alleles a, b, c, d, e, f, g, h and i. These markers were validated on 161 wheat varieties and advanced lines with different Glu-B3 alleles, thus confirming that the markers can be used in marker-assisted breeding for wheat grain processing quality. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. L. H. Wang and X. L. Zhao contributed equally to this study.     

Spelt-specific alleles in HMW glutenin genes from modern and historical European spelt ( Triticum spelta L.)

A partial promoter region of the high-molecular weight (HMW) glutenin genes was studied in two wheat specimens, a 300 year-old spelt (Triticum spelta L.) and an approximately 250 year-old bread wheat (Triticum aestivum L.) from Switzerland. Sequences were compared to a recent Swiss landrace T. spelta ’Oberkulmer.’ The alleles from the historical bread wheat were most similar to those of modern T. aestivum cultivars, whereas in the historical and the recent spelt specific alleles were detected. Pairwise genetic distances up to 0.03 within 200 bp from the HMW Glu-A1-2, Glu-B1-1 and Glu-B1-2 alleles in spelt to the most-similar alleles from bread wheat suggest a polyphyletic origin. The spelt Glu-B1-1 allele, which was unlike the corresponding alleles in bread wheat, was closer related to an allele found in tetraploid wheat cultivars. The results are discussed in context of the origin of European spelt. Received: 22 July 2000 / Accepted: 27 April 2001     

Identification and mapping of pm2026: a recessive powdery mildew resistance gene in an einkorn (Triticum monococcum L.) accession

Triticum monococcum accession TA2026 showed resistance to wheat powdery mildew. To identify the resistance gene and transfer it to common wheat, genetic analysis and molecular mapping were conducted using an F₂ population and derived F₃ families from the cross of TA2026 × M389. The results indicated that TA2026 possessed a recessive powdery mildew resistance gene. This gene was mapped to the terminal portion of chromosome 5A^mL and flanked by SSR marker loci Xcfd39 and Xgwm126. Eight RFLP markers previously mapped to the terminal chromosome 5A^mL were converted into STS markers. Three loci, detected by MAG1491, MAG1493 and MAG1494, the STS markers derived from RFLP probes CDO1312, PSR164 and PSR1201, respectively, were linked to this resistance gene with Xmag1493 only 0.9 cM apart from it. In addition, the STS marker MAG2170 developed from the tentative consensus wheat cDNA encoding the Mlo-like protein identified a locus co-segregating with Xmag1493. This is the first recessive powdery mildew resistance gene identified on chromosome 5A^m, and is temporarily designated pm2026. We have successfully transferred it to a tetraploid background, and this resistance stock will now be used as the bridge parent for its transfer to common wheat.     

Isolation of low-molecular-weight glutenin subunit genes from wild emmer wheat (Triticum dicoccoides)

Three low-molecular-weight glutenin subunit (LMW-GS) genes, designated LMW-Td1, LMW-Td2 and LMW-Td3, were isolated from wild emmer wheat (Triticum dicoccoides), which is the tetraploid progenitor of common wheat (T. aestivum). The complete nucleotide sequence lengths of LMW-Td1, LMW-Td2 and LMW-Td3 are 858, 900 and 1062 bp, respectively. LMW-Td1 and LMW-Td3 can encode proteins with 284 and 352 amino acid residues, respectively, whereas LMW-Td2 is a putative pseudogene due to the presence of 3 inframe stop codons in its C-terminal domain. The deduced protein sequences of the 3 genes share the same typical polypeptide structures with known LMW-GS genes containing 8 cysteines in the mature protein domains. LMW-Td1 was clearly distinguished from all known LMW-GS genes, and considered as a novel LMW-GS gene. Two hydrophobic motifs (i.e. PIIIL and PVIIL) were observed in the repetitive domain of LMW-Td3. Sequence comparison indicates that sequences of the 3 LMW-GS genes from this study are strongly similar to known LMW-GS genes. Our phylogenetic analysis suggests that LMW-Td1 and LMW-Td2 are homologous with genes on chromosome 1A, and LMW-Td3 is closely related to genes on chromosome 1B.     

Genetic transformation of einkorn (<Emphasis Type="Italic">Triticum monococcum L.</Emphasis> ssp. monococcum L.), a diploid cultivated wheat species

Background

Domesticated einkorn (Triticum monococcum L.) is one of the oldest cultivated cereal crops in the world. Its small genome size (~?5.7 GB), low ploidy (2n?=?2x?=?14, A^mA^m) and high genetic polymorphism make this species very attractive for use as a diploid model for understanding the genomics and proteomics of Triticeae. Einkorn, however, is still a recalcitrant monocotyledonous species for the application of modern biotechnologies, including transgenesis. This paper reports the factors that may influence transgene delivery, integration, expression and inheritance in einkorn.

Results

In this study, we report the successful genetic transformation of einkorn using biolistic-mediated DNA delivery. Immature embryo-derived tissues of spring einkorn were bombarded with a plasmid containing the reporter gene GFP (green fluorescent protein) driven by the rice actin promoter (act1) and the selectable bar gene (bialaphos resistance gene) driven by the maize ubiquitin promoter (ubi1). Adjustments to various parameters such as gas pressure, microcarrier size and developmental stage of target tissue were essential for successful transient and stable transformation. Bombarded einkorn tissues are recalcitrant to regenerating plants, but certain modifications of the culture medium have been shown to increase the production of transgenic events. In various experiments, independent transgenic plants were produced at frequencies ranging from 0.0 to 0.6%. Molecular analysis, marker gene expression and herbicide treatment demonstrated that gfp/bar genes were stably integrated into the einkorn genome and successfully inherited over several generations. The transgenes, as dominant loci, segregated in both Mendelian and non-Mendelian fashion due to multiple insertions. Fertile homozygous T₁-T₂ populations of transgenic einkorn that are resistant to herbicides were selected.

Conclusion

To the best of our knowledge, this is the first report of the production of genetically modified einkorn plants. We believe that the results of our research could be a starting point for the application of the current biotechnological-based technologies, such as transgenesis and genome editing, to accelerate comparative functional genomics in einkorn and other cereals.

     

A novel high molecular weight glutenin subunit from Australopyrum retrofractum

We describe the sequence of a gene encoding a high molecular weight glutenin subunit (HMW-GS) expressed in the endosperm of the wheat relative Australopyrum retrofractum. Although the subunit has a similar primary structure to that HMW-GS genes present in other Triticeae species, its N-terminal domain is shorter, its central repetitive domain includes a unique dodecameric motif, and its C-terminal domain contain an extra cysteine residue. A phylogenetic analysis showed that the Glu-W1 gene is neither a true x- nor a true y-type subunit, although it is more closely related to the y-type genes present in the K and E genomes than to any other published HMW-GS gene. All these results indicated that this novel subunit may undergo a special evolutionary process different from other Triticeae species. A flour supplementation experiment showed that the Glu-W1 subunit has a negative effect on dough quality, which might be the result of interaction between the two closely placed cysteine residues in the C-terminal region.     

Cloning and molecular characterization of a novel x-type glutenin gene Glu-D(t)1 from Aegilops tauschii

A novel gene encoding an x-type high molecular weight glutenin subunit (HMW-GS), designated 1Dx1.1 ^t , was isolated from Aegilops tauschii. It is the largest HMW-GS gene reported so far in this species and its product has a slower mobility than that of subunit 1Ax1 in SDS-PAGE. The open reading frame (ORF) of the gene was 2,628 bp, encoding a protein of 874 amino acid residues. Comparisons of amino acid sequences showed that subunit 1Dx1.1^t had high similarity with other 1Dx subunits but also had two unique characteristics. Firstly, a tripeptide of consensus LQE present in the N-terminal domains of other 1Dx subunits was absent from subunit Dx1.1^t. Secondly, three copies of tandem duplications of the tripeptide motif GQQ and a novel tripeptide sequence (GQL) were present in its central repetitive domain. Phylogenetic analysis showed that subunit 1Dx1.1^t clustered with other known 1Dx subunits.     

Structural homology of endosperm high molecular weight glutenin subunits of common wheat (Triticum aestivum L.)

Summary Several high molecular weight endosperm glutenin subunits, coded by genes located on chromosomes 1A, 1B and 1D of common wheat, Triticum aestivum L. em. Thell., were isolated from excised gel segments and subjected to amino acid analysis and peptide mapping; the latter was carried out following a limited digestion with trypsin, chymotrypsin or Staphylococcus aureus — V8 protease. Generally, all high molecular weight glutenins had a similar amino acid composition but several significant differences were observed in some of them. Both analyses revealed that the structural similarity among the various subunits was related to the homology of the genes coding them: subunits coded by homoalleles, i.e., different alleles of the same gene, were most similar; those coded by homoeoalleles, i.e., alleles of homoeologous genes, were less similar; whereas subunits coded either by alleles of different genes of the same gene cluster, or by nonhomoeoalleles of homoeologous clusters, were the least similar. Several small peptides derived from protease digestion of various subunits had a higher than expected staining intensity indicating that small peptide repeats may be interspersed within the glutenin subunits. The evolutionary course of the high molecular weight glutenins is discussed.