Induction of Targeted Deletions in Transgenic Bread Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) Using Customized Meganuclease

Biotechnologies offer breeders good opportunities for breakthrough genetic improvements of bread wheat, one of mankind’s main food crops. Since the production of the first transgenic wheat, one of the major concerns has been the removal of selective markers, first because of societal concerns about the antibiotic resistance of some of these genes, and second because removal of a selective marker was the first step toward retransformation using the same selection system. Site-directed nucleases are enzymes that cut genomic DNA in vivo at predefined sites. Among them, meganucleases cut DNA at predefined, long DNA (up to 24 nt) sites, thereby enabling single cuts on large genomes including the bread wheat genome (17 Gbp). In this paper, we describe for the first time the use of a customized meganuclease to cut wheat DNA in vivo. We show that double cuts provoked the deletion of previously inserted DNA cassettes containing the DsRed reporter gene, and that in many cases, the meganuclease target site was correctly reconstituted, offering opportunities for subsequent insertion of stacked transgenes to replace the gene of selection. Moreover, perfect deletions were observed not only in the callus after transient expression of the meganucleases, but also in T0 transgenic wheat after stable retransformation with the meganuclease. Future prospects for the removal of selective markers and transgene stacking are discussed.     

The endophytic fungi from wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

In order to study the species composition of endophytes from wheat healthy plants in Buenos Aires Province (Argentina) and to determine their infection frequencies from leaves, stems, glumes and grains, wheat plants were collected from five cultivars at five growth stages from crop emergence to harvest. A total of 1,750 plant segments (leaves, stems, glumes and grains) were processed from the five wheat cultivars at five growth stages, and 722 isolates of endophytic fungi recovered were identified as 30 fungal genera. Alternaria alternata, Cladosporium herbarum, Epicoccum nigrum, Cryptococcus sp., Rhodotorula rubra, Penicillium sp. and Fusarium graminearum were the fungi that showed the highest colonization frequency (CF%) in all the tissues and organs analysed. The number of taxa isolated was greater in the leaves than those in the other organs analysed.     

Analysis of the Distribution of <Emphasis Type="Italic">Triticum timopheevii</Emphasis> Zhuk. Genetic Material in Common Wheat Varieties (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The database of the world gene pool of wheat was scanned by pedigree and the participation of genetic material from T. timopheevii in the creation of 3088 varieties of common wheat was established. The spatial and temporal dynamics of the propagation of these varieties was studied. Using the analysis of pedigrees, a diversity of T. timopheevii donors was studied. The specificity of donors of the genetic material T. timopheevii for the regions of wheat breeding was established. The main source of resistance genes for most varieties is accession D-357-1 from the Georgian variety-population of Zanduri. This significantly reduces the diversity of the genetic material of T. timopheevii used in wheat breeding. In 369 varieties and 184 lines, the genes for resistance to pathogens from T. timopheevii were identified. The genes of T. timopheevii are distributed mainly in winter varieties, as well as spring varieties sown in autumn. The value of donors as sources of T. timopheevii genes is ambiguous, despite the fact that most of them come from the same D-357-1 accession. The Sr36 gene is most commonly found in the United States, Western Europe, and Australia; it was transferred from the Wisconsin-245 line through Arthur or TP-114-1965a. The Pm6 gene is distributed in Western Europe; it was transferred from the pre-breeding line Wisconsin 245/5*Cappelle-Desprez//Hybrid- 46/Cappelle Desprez. The gene Lr18 is more common in the United States; it was transmitted by the Blueboy or Vogel 5 varieties from the Coker-55-9 line. The extremely limited set of genes for resistance to pathogens from T. timopheevii used in commercial varieties and the specificity of their geographical distribution are possibly associated with the uniqueness of the G subgenome and plasmon in this species, its low potential for plasticity, and tolerance to drought. In addition, the imperfection of the methods of pre-breeding and recombination breeding prevents the elimination in translocation of close linkage of target genes with undesirable ones.     

Overexpression of <Emphasis Type="Italic">AtHDG11</Emphasis> enhanced drought tolerance in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Drought is one of the major abiotic stresses restricting the yield of wheat (Triticum aestivum L.). Breeding wheat varieties with drought tolerance is an effective and durable way to fight against drought. Here we reported introduction of AtHDG11 into wheat via Agrobacterium-mediated transformation and analyzed the morphological and physiological characteristics of T2 generation transgenic lines under drought stress. With drought treatment for 30 days, transgenic plants showed significantly improved drought tolerance. Compared with controls, the transgenic lines displayed lower stomatal density, lower water loss rate, more proline accumulation and increased activities of catalase and superoxide dismutase. Without irrigation after booting stage, the photosynthetic parameters, such as net photosynthesis rate, water use efficiency and efficiency of excitation energy, were increased in transgenic lines, while transpiration rate was decreased. Moreover, the kernel yield of transgenic lines was also improved under drought condition. Taken together, our data demonstrate that AtHDG11 has great potential in genetic improvement of drought tolerance of wheat.     

Structure and expression of the <Emphasis Type="Italic">TaGW7</Emphasis> in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

OsGW7 (also known as OsGL7) is homologous to the Arabidopsis thaliana gene that encodes LONGIFOLIA protein, which regulates cell elongation, and is involved in regulating grain length in rice. However, our knowledge on its ortholog in wheat, TaGW7, is limited. In this study, we identified and mapped TaGW7 in wheat, characterized its nucleotide and protein structures, predicted the cis-elements of its promoter, and analysed its expression patterns. The GW7 orthologs in barley (HvGW7), rice (OsGW7), and Brachypodium distachyon (BdGW7) were also identified for comparative analyses. TaGW7 mapped onto the short arms of group 2 chromosomes (2AS, 2BS, and 2DS). Multiple alignments indicated GW7 possesses five exons and four introns in all but two of the species analysed. An exon–intron junction composed of introns 3–4 and exons 4–5 was highly conserved. GW7 has a conserved domain (DUF 4378) and two neighbouring low complexity regions. GW7 was mainly expressed in wheat spikes and stems, in barley seedling crowns, and in rice anthers and embryo-sacs during early development. Drought and heat significantly increased and decreased GW7 expression in wheat, respectively. In barley, GW7 was significantly down-regulated in paleae and awns but up-regulated in seeds under drought treatment and down-regulated under Fusarium and stem rust inoculation. In rice, OsGW7 expression differed significantly under drought treatments. Collectively, these results provide insights into GW7 structure and expression in wheat, barley and rice. The GW7 sequence structure and expression data are the foundation for manipulating GW7 and uncovering its roles in plants.     

A search for high-molecular-weight subunits of glutenin from <Emphasis Type="Italic">Triticum timopheevi</Emphasis> Zhuk. in the lines of common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L. <Emphasis Type="Italic">Triticum timopheevi</Emphasis> Zhuk.)

The study is a continuation of investigation of prolamins in brown rust-resistant introgressive lines of common wheat, produced with participation of Triticum timopheeevi Zhuk. [1]. Two wheat lines with a substitution of the Glu-1 loci of T. timopheevi were identified. Line 684 had high-molecular-weight glutenin subunits encoded by 1Ax, as well as by 1Ay gene, which was silent in commercial lines. It was demonstrated that line 684 could serve as a source of the Glu-A _t ¹ locus. Line 186 carried the Glu-B1/Glu-G1 substitution. Comparative analysis of storage proteins from the introgression lines of common wheat Triticum aestivum L. with those from parental forms demonstrated polymorphism among the lines, resulted from natural varietal polymorphism, and introgression of the Glu-3 and Gli-1 loci from the genome of T. timopheevi.     

The Genome of Bread Wheat <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.: Unique Structural and Functional Properties

Polyploidy is the major mechanism of speciation in flowering plants. All genomes of ancient species that are the progenitors of extant plant species experienced polyploidization. Three consecutive stages of polyploidization, i.e., ancient polyploidization, tetra-, and hexaploidization, resulted in the emergence of modern allohexaploid bread wheat Triticum aestivum L. with the BBAADD genome. Polyploidization and subsequent stabilization of the polyploid genome of T. aestivum led, on one hand, to cytological diploidization and, on the other hand, to structural and functional asymmetry of its three subgenomes. In recent years, there has been a sharp increase in the data accumulation on the origin and structure of the bread wheat genomes a result of analysis of genomes and transcripomes of natural and synthetic wheats using modern mapping and sequencing methods. This review provides up-to-date information on the peculiarities of the T. aestivum genome reorganization, which affected its structure and functioning.     

Characteristics of Thiol:Protein Disulfide Oxidoreductase from Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) Grain

Biochemical properties of a homogenous preparation of thiol:protein disulfide oxidoreductase (TPDO, EC 1.8.4.2) isolated for the first time from mature wheat (Triticum aestivum L.) grain were studied. According to polyacrylamide gel electrophoresis data, the molecular weight of TPDO is around 167 kD, the enzyme consisting of two subunits of 77 and 73 kD, which differentiates TPDO from known enzymes of SH/SS-metabolism of wheat caryopses. In substrate specificity and enzymatic characteristics (pH and temperature optima) TPDO is similar to analogous enzymes of animal tissues. Inhibition of disulfide reductase activity by alkylating agents and heavy metal ions suggests the participation of active center SH-groups in the catalytic act and classes the enzyme as a member of the thioredoxin superfamily. The SS-reductase reduces aggregating capacity of acetic acid-soluble fraction of wheat storage proteins. The proposed physiological role of TPDO is participation in creation and regulation of SH/SS-status of wheat endosperm proteins and formation of the rheological properties of gluten.__________Translated from Biokhimiya, Vol. 70, No. 8, 2005, pp. 1130– 1136.Original Russian Text Copyright © 2005 by Osipova, Permyakov, Mitrofanova, Dudareva, Trufanov.     

Cloning and characterization of microRNAs from wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Background  

MicroRNAs (miRNAs) are a class of small, non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition. So far, identification of miRNAs has been limited to a few model plant species, such as Arabidopsis, rice and Populus, whose genomes have been sequenced. Wheat is one of the most important cereal crops worldwide. To date, only a few conserved miRNAs have been predicted in wheat and the computational identification of wheat miRNAs requires the genome sequence, which is unknown.     

Evidence of intralocus recombination at the <Emphasis Type="Italic">Glu-3</Emphasis> loci in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Key message

Recombination at the Glu-3 loci was identified, and strong genetic linkage was observed only between the amplicons representing i-type and s-type genes located, respectively, at the Glu-A3 and Glu-B3 loci.

Abstract

The low-molecular weight glutenin subunits (LMW-GSs) are one of the major components of wheat seed storage proteins and play a critical role in the determination of wheat end-use quality. The genes encoding this class of proteins are located at the orthologous Glu-3 loci (Glu-A3, Glu-B3, and Glu-D3). Due to the complexity of these chromosomal regions and the high sequence similarity between different LMW-GS genes, their organization and recombination characteristics are still incompletely understood. This study examined intralocus recombination at the Glu-3 loci in two recombinant inbred line (RIL) and one doubled haploid (DH) population, all segregating for the Glu-A3, Glu-B3, and Glu-D3 loci. The analysis was conducted using a gene marker system that consists of the amplification of the complete set of the LMW-GS genes and their visualization by capillary electrophoresis. Recombinant marker haplotypes were detected in all three populations with different recombination rates depending on the locus and the population. No recombination was observed between the amplicons representing i-type and s-type LMW-GS genes located, respectively, at the Glu-A3 and Glu-B3 loci, indicating tight linkage between these genes. Results of this study contribute to better understanding the genetic linkage and recombination between different LMW-GS genes, the structure of the Glu-3 loci, and the development of more specific molecular markers that better represent the genetic diversity of these loci. In this way, a more precise analysis of the contribution of various LMW-GSs to end-use quality of wheat may be achieved.

     

Genetic architecture of seed longevity in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The deterioration in the quality of ex situ conserved seed over time reflects a combination of both physical and chemical changes. Intraspecific variation for longevity is, at least in part, under genetic control. Here, the grain of 183 bread wheat accessions maintained under low-temperature storage at the IPK-Gatersleben genebank over some decades have been tested for their viability, along with that of fresh grain subjected to two standard artificial ageing procedures. A phenotype–genotype association analysis, conducted to reveal the genetic basis of the observed variation between accessions, implicated many regions of the genome, underling the genetic complexity of the trait. Some, but not all, of these regions were associated with variation for both natural and experimental ageing, implying some non-congruency obtains between these two forms of testing for longevity. The genes underlying longevity appear to be independent of known genes determining dormancy and pre-harvest sprouting.     

QTL mapping of flag leaf-related traits in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Key message

QTL controlling flag leaf length, flag leaf width, flag leaf area and flag leaf angle were mapped in wheat.

Abstract

This study aimed to advance our understanding of the genetic mechanisms underlying morphological traits of the flag leaves of wheat (Triticum aestivum L.). A recombinant inbred line (RIL) population derived from ND3331 and the Tibetan semi-wild wheat Zang1817 was used to identify quantitative trait loci (QTLs) controlling flag leaf length (FLL), flag leaf width (FLW), flag leaf area (FLA), and flag leaf angle (FLANG). Using an available simple sequence repeat genetic linkage map, 23 putative QTLs for FLL, FLW, FLA, and FLANG were detected on chromosomes 1B, 2B, 3A, 3D, 4B, 5A, 6B, 7B, and 7D. Individual QTL explained 4.3–68.52% of the phenotypic variance in different environments. Four QTLs for FLL, two for FLW, four for FLA, and five for FLANG were detected in at least two environments. Positive alleles of 17 QTLs for flag leaf-related traits originated from ND3331 and 6 originated from Zang1817. QTLs with pleiotropic effects or multiple linked QTL were also identified on chromosomes 1B, 4B, and 5A; these are potential target regions for fine-mapping and marker-assisted selection in wheat breeding programs.

     

Assessment of genetic diversity among Syrian durum (<Emphasis Type="Italic">Triticum</Emphasis> ssp. <Emphasis Type="Italic">durum</Emphasis>) and bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) using SSR markers

Genetic diversity among 49 wheat varieties (37 durum and 12 bread wheat) was assayed using 32 microsatellites representing 34 loci covering almost the whole wheat genome. The polymorphic information content (PIC) across the tested loci ranged from 0 to 0.88 with average values of 0.57 and 0.65 for durum and bread wheat respectively. B-genome had the highest mean number of alleles (10.91) followed by A genome (8.3) whereas D genome had the lowest number (4.73). The correlation between PIC and allele number was significant in all genome groups accounting for 0.87, 074 and 0.84 for A, B and D genomes respectively, and over all genomes, the correlation was higher in tetraploid (0.8) than in hexaploid wheat varieties (0.5). The cluster analysis discriminated all varieties and clearly divided the two ploidy levels into two separate clusters that reflect the differences in genetic diversity within each cluster. This study demonstrates that microsatellites markers have unique advantages compared to other molecular and biochemical fingerprinting techniques in revealing the genetic diversity in Syrian wheat varieties that is crucial for wheat improvement.     

A <Emphasis Type="Italic">Pseudo-Response Regulator</Emphasis> is misexpressed in the photoperiod insensitive <Emphasis Type="Italic">Ppd-D1a</Emphasis> mutant of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Ppd-D1 on chromosome 2D is the major photoperiod response locus in hexaploid wheat (Triticum aestivum). A semi-dominant mutation widely used in the “green revolution” converts wheat from a long day (LD) to a photoperiod insensitive (day neutral) plant, providing adaptation to a broad range of environments. Comparative mapping shows Ppd-D1 to be colinear with the Ppd-H1 gene of barley (Hordeum vulgare) which is a member of the pseudo-response regulator (PRR) gene family. To investigate the relationship between wheat and barley photoperiod genes we isolated homologues of Ppd-H1 from a ‘Chinese Spring’ wheat BAC library and compared them to sequences from other wheat varieties with known Ppd alleles. Varieties with the photoperiod insensitive Ppd-D1a allele which causes early flowering in short (SD) or LDs had a 2 kb deletion upstream of the coding region. This was associated with misexpression of the 2D PRR gene and expression of the key floral regulator FT in SDs, showing that photoperiod insensitivity is due to activation of a known photoperiod pathway irrespective of day length. Five Ppd-D1 alleles were found but only the 2 kb deletion was associated with photoperiod insensitivity. Photoperiod insensitivity can also be conferred by mutation at a homoeologous locus on chromosome 2B (Ppd-B1). No candidate mutation was found in the 2B PRR gene but polymorphism within the 2B PRR gene cosegregated with the Ppd-B1 locus in a doubled haploid population, suggesting that insensitivity on 2B is due to a mutation outside the sequenced region or to a closely linked gene. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. J. Beales and A. Turner contributed equally to the work.     

Flow karyotyping and chromosome sorting in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Previously, we reported on the development of procedures for chromosome analysis and sorting using flow cytometry (flow cytogenetics) in bread wheat. That study indicated the possibility of sorting large quantities of intact chromosomes, and their suitability for analysis at the molecular level. However, due to the lack of sufficient differences in size between individual chromosomes, only chromosome 3B could be sorted into a high-purity fraction. The present study aimed to identify wheat stocks that could be used to sort other chromosomes. An analysis of 58 varieties and landraces demonstrated a remarkable reproducibility and sensitivity of flow cytometry for the detection of numerical and structural chromosome changes. Changes in flow karyotype, diagnostic for the presence of the 1BL·1RS translocation, have been found and lines from which translocation chromosomes 5BL·7BL and 4AL·4AS-5BL could be sorted have been identified. Furthermore, wheat lines have been identified which can be used for sorting chromosomes 4B, 4D, 5D and 6D. The ability to sort any single arm of the hexaploid wheat karyotype, either in the form of a ditelosome or a isochromosome, has also been demonstrated. Thus, although originally considered recalcitrant, wheat seems to be suitable for the development of flow cytogenetics and the technology can be applied to the physical mapping of DNA sequences, the targeted isolation of molecular makers and the construction of chromosome- and arm-specific DNA libraries. These approaches should facilitate the analysis of the complex genome of hexaploid bread wheat.     

Asymmetric somatic hybridization between wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) and <Emphasis Type="Italic">Avena sativa</Emphasis> L.

Protoplasts from cell suspensions of young-embryo-derived calli, whichwere non- regenerable for long-term subculture and protoplasts from embryogenic calli with the regeneration capacity of 75% of the same wheat Jinan 177, were mixed as recipient. Protoplasts from embryogenic calli of Avena sativa (with the regeneration capacity of less than 10%) irradiated with UV at an intensity of300 μW/cm2 for 30 s, 1 min, 2 min, 3 min, 5 min were used as the donor. Protoplasts of the recipient and the donor were fused by PEG method. Many calli and normal green plants were regenerated at high frequency, and were verified as somatic hybrids by chromosome counting, isozyme, 5S rDNA spacer sequence analysis and GISH (genomic in situ hybridization). Fusion combination between protoplasts either from the cell suspensions or from the calli and UV-treated Avena sativa protoplasts could not regenerate green plants.