Mapping and characterization of major QTL for spike traits in common wheat.

The spike traits of wheat can directly affect yield. F2 and F2:3 lines derived from the cross of the multi-spikelet female 10-A and the uni-spikelet male BE89 were used to detect QTLs for spike length (SL), total spikelet number per spike (TSS), kernel number per spike (KNS) and thousand-kernel weight (TKW) in four different environments. A total of 1098 SNP and 5 SSR were used to construct genetic map of 2398.1 cM with the average distance of 2.2 cM between markers. A total of 11 QTLs were identified for spike traits, including three QTLs for SL, five QTLs for TSS, two QTLs for KNS and one QTL for TKW. The QTLs mapped to chromosomes 2D, 4A, 6A, 7A and 7B explained 8.2-37.8% of the phenotypic variation in single environment. The major QTL confidence interval with distance of 0.5 cM was located on chromosome 4A and detected in multiple environments, which can explain more than 30% of the phenotypic variation for SL, TSS and KNS. Combining IWGSC RefSeq v1.0 and RNA-seq data for 10-A and BE89, we identified 16 genes expressed on spike or grain in four QTL regions. These findings provide insights into improving wheat yield through increasing spikletes in wheat, particularly through the use of the multi-spikelet female 10-A for breeding.

Transcriptional reference map of hormone responses in wheat spikes.

BACKGROUND: Phytohormones are key regulators of plant growth, development, and signalling networks involved in responses to diverse biotic and abiotic stresses. Transcriptional reference maps of hormone responses have been reported for several model plant species such as Arabidopsis thaliana, Oryza sativa, and Brachypodium distachyon. However, because of species differences and the complexity of the wheat genome, these transcriptome data are not appropriate reference material for wheat studies. RESULTS: We comprehensively analysed the transcriptomic responses in wheat spikes to seven phytohormones, including indole acetic acid (IAA), gibberellic acid (GA), abscisic acid (ABA), ethylene (ET), cytokinin (CK), salicylic acid (SA), and methyl jasmonic acid (MeJA). A total of 3386 genes were differentially expressed at 24 h after the hormone treatments. Furthermore, 22.7% of these genes exhibited overlapping transcriptional responses for at least two hormones, implying there is crosstalk among phytohormones. We subsequently identified genes with expression levels that were significantly and differentially induced by a specific phytohormone (i.e., hormone-specific responses). The data for these hormone-responsive genes were then compared with the transcriptome data for wheat spikes exposed to biotic (Fusarium head blight) and abiotic (water deficit) stresses. CONCLUSION: Our data were used to develop a transcriptional reference map of hormone responses in wheat spikes.

Re-acquisition of the brittle rachis trait via a transposon insertion in domestication gene Q during wheat de-domestication.

De-domestication is a unique evolutionary process during which crops re-acquire wild-like traits to survive and persist in agricultural fields without the need for human cultivation. The re-acquisition of seed dispersal mechanisms is crucial for crop de-domestication. Common wheat is an important cereal crop worldwide. Tibetan semi-wild wheat is a potential de-domesticated common wheat subspecies. However, the crucial genes responsible for its brittle rachis trait have not been identified. Genetic mapping, functional analyses and phylogenetic analyses were completed to identify the gene associated with Qbr.sau-5A, which is a major locus for the brittle rachis trait of Tibetan semi-wild wheat. The cloned Qbr.sau-5A gene is a new Q allele (Qt ) with a 161-bp transposon insertion in exon 5. Although Qt is expressed normally, its encoded peptide lacks some key features of the APETALA2 family. The abnormal functions of Qt in developing wheat spikes result in brittle rachises. Phylogenetic and genotyping analyses confirmed that Qt originated from Q in common wheat and is naturally distributed only in Tibetan semi-wild wheat populations. The identification of Qt provides new evidence regarding the origin of Tibetan semi-wild wheat, and new insights into the re-acquisition of wild traits during crop de-domestication.

Alternative splicing results in a lack of starch synthase IIa-D in Chinese wheat landrace.

We evaluated the SGP-1 protein composition of 368 Chinese wheat landraces using SDS-PAGE. The SGP-D1 null type was identified in three accessions (Xiaoqingmang, Pushanbamai, and P119). An 18-bp deletion and 9-bp variation were found at the junction region of the 7th intron and 8th exon, leading to deletion of the intron-exon junction recognition site AG when aligned the 8261-bp DNA sequence of TaSSIIa-D in Pushanbamai with that of Chinese Spring. Four cDNA types with mis-spliced isoforms were subsequently detected through amplification of TaSSIIa-D cDNAs. Among these, nine type II cDNAs with a 16-bp deletion in the 8th exon were detected, indicating that the major transcriptional pattern of TaSSIIa in Pushanbamai is type II. In the type IV cDNA, a 97-bp sequence remains undeleted in the end of the 5th exon. The amylose content in Pushanbamai was significantly higher than that in all control lines under field conditions, which suggested that deletion of SGP-D1 has an efficient impact on amylose content. As the TaSSIIa gene plays an important role in regulating the content of amylose, it is anticipated that these natural variants of TaSSIIa-D will provide useful resources for quality improvement in wheat.

Fusarium graminearum ATP-Binding Cassette Transporter Gene FgABCC9 Is Required for Its Transportation of Salicylic Acid, Fungicide Resistance, Mycelial Growth and Pathogenicity towards Wheat.

ATP-binding cassette (ABC) transporters hydrolyze ATP to transport a wide range of substrates. Fusarium graminearum is a major causal agent of Fusarium head blight, which is a severe disease in wheat worldwide. FgABCC9 (FG05_07325) encodes an ABC-C (ABC transporter family C) transporter in F. graminearum, which was highly expressed during the infection in wheat and was up-regulated by the plant defense hormone salicylic acid (SA) and the fungicide tebuconazole. The predicted tertiary structure of the FgABCC9 protein was consistent with the schematic of the ABC exporter. Deletion of FgABCC9 resulted in decreased mycelial growth, increased sensitivity to SA and tebuconazole, reduced accumulation of deoxynivalenol (DON), and less pathogenicity towards wheat. Re-introduction of a functional FgABCC9 gene into ΔFgABCC9 recovered the phenotypes of the wild type strain. Transgenic expression of FgABCC9 in Arabidopsis thaliana increased the accumulation of SA in its leaves without activating SA signaling, which suggests that FgABCC9 functions as an SA exporter. Taken together, FgABCC9 encodes an ABC exporter, which is critical for fungal exportation of SA, response to tebuconazole, mycelial growth, and pathogenicity towards wheat.

Transposon insertion resulted in the silencing of Wx-B1n in Chinese wheat landraces.

KEY MESSAGE: A novel Wx-B1 allele was characterized; a transposon insertion resulted in the loss of its function, which is different from the previously reported gene silencing mechanisms at the Wx-B1 locus. The waxy protein composition of 53 Chinese wheat landraces was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis; of these, 10 did not show the expression of Wx-A1 (four accession) or Wx-B1 (six accessions) protein. The results of molecular marker detection revealed that the Wx-B1 allele (Wx-B1n) showed normal expression, inconsistent with the findings of SDS-PAGE for the Xiaobaipi accession. Further cloning of the 9160-bp region covering the Wx-B1 coding region and 3'-downstream region revealed that a 2178-bp transposon fragment had been inserted at 2462 bp within the tenth exon of Wx-B1n ORF, leading to the absence of Wx-B1 protein. Sequence analysis indicated that the insertion possessed the structural features of invert repeat and target repeat elements, we deduced that it was a transposon. Further PCR analysis revealed that this fragment had moved, but not copied itself, from 3B chromosome to the current location in Wx-B1n. Therefore, the reason for the inactivation of Wx-B1n was considerably different from those for the inactivation of Wx-B1b, Wx-B1k, and Wx-B1m; to our knowledge, this kind of structural mutation has never been reported in Wx-B1 alleles. This novel allele is interesting, because it was not associated with the deletion of other quality-related genes included in the 67 kb region lost with the common null allele Wx-B1b. The null Wx-B1n might be useful for investigating gene inactivation and expression as well as for enriching the genetic resource pool for the modification of the amylose/amylopectin ratio, thereby improving wheat quality.

Identification and characterization of genes on a single subgenome in the hexaploid wheat (Triticum aestivum L.) genotype 'Chinese Spring'.

Gene loss during the formation of hexaploid bread wheat has been repeatedly reported. However, our knowledge on genome-wide analysis of the genes present on a single subgenome (SSG) in bread wheat is still limited. In this study, by analysing the 'Chinese Spring' chromosome arm shotgun sequences together with high-confidence gene models, we detected 433 genes on a SSG. Greater gene loss was observed in A and D subgenomes compared with B subgenome. More than 79% of the orthologs for these SSG genes were detected in diploid and tetraploid relatives of hexaploid wheat. Unexpectedly, no bias in expression breadth or in the distribution patterns of GO (gene ontology) terms for these genes was detected among the high-confidence genes. Further, network and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses indicated that most of these genes were not functionally related to each other. Interestingly, 30.7% of these SSG genes were most highly expressed in root, showing biased distribution given the distribution of the whole high-confidence genes. Collectively, these results facilitate our understanding of the loss of the genes that were retained in a SSG during the formation of hexaploid wheat.

Identification of quantitative trait loci for seedling root traits from Tibetan semi-wild wheat (Triticum aestivum subsp. tibetanum).

As a primitive hexaploid wheat resource distributed only in Tibet, Tibetan semi-wild wheat (Triticum aestivum subsp. tibetanum Shao) possesses unique characteristics that could be exploited in wheat breeding programs. Its good root system could offer a stable platform for above-ground components. To detect possible excellent locus for root traits from Tibetan semi-wild wheat, we identified QTLs for root traits using a recombinant inbred line population derived from a cross between Tibetan semi-wild wheat Q1028 and Zhengmai 9023. A total of 15 QTLs on eight chromosomes were detected, including four major QTLs, QMrl.sau-7B, QTrl.sau-4B, QAd.sau-7A, and QSa.sau-4B. The phenotypic variation explained by each of these QTLs ranges from 5.67% to 16.68%. Positive alleles of six QTLs were derived from Q1028. Several novel QTLs for root traits were identified. In addition, significant correlations were detected amongst root traits and agronomic traits. Taken together, these results suggest that Tibetan semi-wild wheat and the newly identified novel QTLs could be useful in future breeding programs.

A super twin T-DNA vector that allows independent gene expression during Agrobacterium-mediated transformation.

In this study, we designed and constructed a super twin T-DNA vector (pTRIDT313-g) containing two independent T-DNA cassettes-one for the selection gene Hyg and the other for the target gene Gus-to produce marker-free transgenic lines. The resulting vector was transformed into tobacco, and polymerase chain reaction (PCR) analysis showed four types of gene combinations in the T1 and T2 generations: Gus only, Hyg only, Gus+Hyg, and untransformed lines. The intermediate region from the T-DNA of the right border of Hyg to the left border of Gus in the Hyg and Gus lines was not amplified. Genome walking confirmed that the Hyg and Gus T-DNA cassettes were independently inserted in different regions of the tobacco genome. Thus, the two T-DNA cassettes were integrated randomly as independent loci into the tobacco genome. The results of reverse transcription-PCR indicated that Hyg could normally be expressed in the roots, stems, and leaves of transgenic lines, and the resistance test showed that all Hyg transgenic lines could grow in the presence of 50mg/L hygromycin. All Gus transgenic lines showed obvious blue coloration in enzyme activity tests, indicating that the Gus gene could be normally expressed in all the lines. Therefore, the super twin T-DNA vector (pTRIDT313-g) exhibits independent integration, heredity, and normal gene function from two T-DNA cassettes. This vector could be a useful and valuable tool in the production of marker-free transgenic lines.

Structure and expression of phosphoglucan phosphatase genes of Like Sex Four1 and Like Sex Four2 in barley.

Phosphoglucan phosphatases (Like-SEX4 1 and 2; LSF1 and LSF2) were reported to play roles in starch metabolism in leaves of Arabidopsis. In this study, we identified and mapped the LSF1 and LSF2 genes in barley (HvLSF1 and HvLSF2), characterized their gene and protein structures, predicted the cis-elements of their promoters, and analysed their expression patterns. HvLSF1 and HvLSF2 were mapped on the long arm of chromosome 1H (1HL) and 5H (5HL), respectively. Our results revealed varied exon-intron structures and conserved exon-intron junctions in both LSF1 and LSF2 from a range of analysed species. Alignment of protein sequences indicated that cTP and CT domains are much less varied than the functional domains (PDZ, DPS and CBM48). LSF2 was mainly expressed in anthers of barley and rice, and in leaf of Arabidopsis. LSF1 was mainly expressed in endosperm of barley and leaf of Arabidopsis and rice. The expression of LSF1 exhibited a diurnal pattern in rice only and that of LSF2 in both rice and Arabidopsis. Of the investigated stresses, only cold stress significantly reduced expression level of LSF1 and LSF2 in barley and LSF2 in Arabidopsis at late stages of the treatments. While heat treatment significantly decreased expression levels of LSF1 at middle stage (4 h) of a treatment in Arabidopsis only. The strong relationships detected between LSF2 and starch excess4 (SEX4), glucan, water dikinases or phosphoglucan, water dikinases were identified and discussed. Taken together, these results provide information of genetic manipulation of LSF1 and LSF2, especially in monocotyledon and further elucidate their regulatory mechanism in plant development.

Structure and expression analysis of genes encoding ADP-glucose pyrophosphorylase large subunit in wheat and its relatives.

ADP-glucose pyrophosphorylase (AGP), which consists of two large subunits (AGP-L) and two small subunits (AGP-S), controls the rate-limiting step in the starch biosynthetic pathway. In this study, a full-length open reading frame (ORF) of AGP-L gene (named as Agp2) in wheat and a series of Agp2 gene sequences in wheat relatives were isolated. The coding region of Agp2 contained 15 exons and 14 introns including a full-length ORF of 1566 nucleotides, and the deduced protein contained 522 amino acids (57.8 kDa). Generally, the phylogenetic tree of Agp2 indicated that sequences from A- and D-genome donor species were most similar to each other and sequences from B-genome donor species contained more variation. Starch accumulation and Agp2 expression in wheat grains reached their peak at 21 and 15 days post anthesis (DPA), respectively.

Inheritance and Molecular Mapping of an All-Stage Stripe Rust Resistance Gene Derived from the Chinese Common Wheat Landrace "Yilongtuomai".

Yellow or stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a devastating foliar disease that affects common wheat (Triticum aestivum L.) around the world. In China, common wheat landraces are potential sources of disease and abiotic stress resistance genes for wheat improvement. Yilongtuomai (YL), a wheat landrace from Yilong County, Sichuan Province, shows high levels of resistance against most Chinese Pst races. In this study, the resistance of YL to stripe rust disease was examined in detail. Parent strains, YL and Taichung 29, a variety susceptible to Pst race CYR32, and their F1, F2, and F2:3 offspring, were inoculated with CYR32 during the seedling stage in the field or adult-plant stage in the greenhouse. Results indicated that resistance to CYR32 in YL is conferred by a single dominant gene, designated YrYL The segregating F2 population (352 plants), was analyzed in terms of its resistance locus using simple sequence repeats (SSRs), resistance gene analog polymorphisms (RGAPs), and sequence-related amplified polymorphism (SRAP). A linkage group of 6 SSRs, 2 RGAPs, and 1 SRAP was constructed for the YrYL gene. Using the identified SSRs associated with physical mapping of RGAP using Chinese Spring nullisomic-tetrasomic stocks, the YrYL gene was localized to the short arm of chromosome 7D. The gene was flanked by 1 SSR marker, Xbarc92, and 1 RGAP marker, CLRRfor/Ptokin4, at genetic distances of 5.35 and 9.86 cM, respectively. The YrYL gene was compared to other stripe rust resistance genes reported on chromosome 7D by evaluating its reaction patterns to CYR32 and its pedigree relationship. Our results suggest that the YrYL gene is a new stripe rust resistance gene.

Identification of genes bordering breakpoints of the pericentric inversions on 2B, 4B, and 5A in bread wheat (Triticum aestivum L.).

Chromosome translocation is an important driving force in shaping genomes during evolution. Detailed knowledge of chromosome translocations in a given species and its close relatives should increase the efficiency and precision of chromosome engineering in crop improvement. To identify genes flanking the breakpoints of translocations and inversions as a step toward identifying breakpoints in bread wheat, we systematically analysed genes in the Brachypodium genome against wheat survey sequences and bin-mapped ESTs (expressed sequence tags) derived from the hexaploid wheat genotype 'Chinese Spring'. In addition to those well-known translocations between group 4, 5, and 7 chromosomes, this analysis identified genes flanking the three pericentric inversions on chromosomes 2B, 4B, and 5A. However, numerous chromosomal rearrangements reported in early studies could not be confirmed. The genes flanking the breakpoints reported in this study are valuable for isolating these breakpoints.

Characterization and expression analysis of WOX2 homeodomain transcription factor in Aegilops tauschii.

The WUSCHEL (WUS)-related homeobox (WOX) gene family coordinates transcription during the early phases of embryogenesis. In this study, a putative WOX2 homolog was isolated and characterized from Aegilops tauschii, the donor of D genome of Triticum aestivum. The sequence consisted of 2045 bp, and contained an open reading frame (ORF), encoded 322 amino acids. The predicted protein sequence contained a highly conserved homeodomain and the WUS-box domain, which is present in some members of the WOX protein family. The full-length ORF was subcloned into prokaryotic expression vector pET-30a, and an approximately 34-kDa protein was expressed in Escherichia coli BL21 (DE3) cells with IPTG induction. The molecular mass of the expressed protein was identical to that predicted by the cDNA sequence. Phylogenetic analysis suggested that Ae. tauschii WOX2 is closely related to the rice and maize orthologs. Quantitative PCR analysis showed that WOX2 from Ae. tauschii was primarily expressed in the seeds; transcription increased during seed development and declined after the embryos matured, suggesting that WOX2 is associated with embryo development in Ae. tauschii.

Characterization of high-molecular-weight glutenin subunits from Eremopyrum bonaepartis and identification of a novel variant with unusual high molecular weight and altered cysteine residues.

We characterized two high-molecular-weight glutenin subunit (HMW-GS) variants from Eremopyrum bonaepartis, determined their complete open reading frames, and further expressed them in a bacterial system. The variants have many novel structural features compared with typical subunits encoded by Glu-1 loci: 1Fx3.7 and 1Fy1.5 exhibit hybrid properties of x- and y-type subunits. In addition, unusual molecular mass and altered number and distribution of cysteine residues were unique features of HMW-GSs encoded by Glu-F1 from E. bonaepartis. The mature 1Fx3.7 subunit has a full length of 1,223 amino acid residues, making it the largest subunit found thus far, while 1Fy1.5 is just 496 residues. In addition, the mutated PGQQ repeat motif was found in the repetitive region of 1Fx3.7. Although it has a similar molecular mass to that previously reported for 1Dx2.2, 1Dx2.2* and 1S(sh)x2.9 subunits, 1Fx3.7 appears to have had a different evolutionary history. The N-terminal and repetitive regions have a total of four additional cysteine residues, giving 1Fx3.7 a total of eight cysteines, while 1Fy1.5 has only six cysteines because the GHCPTSPQQ nonapeptide at the end of the repetitive region is deleted. With its extra cysteine residues and the longest repetitive region, features that are relevant to good wheat quality, the 1Fx3.7 subunit gene could be an excellent candidate for applications in wheat quality improvement.

Conserved structure and varied expression reveal key roles of phosphoglucan phosphatase gene starch excess 4 in barley.

As one of the phosphoglucan phosphatases, starch excess 4 (SEX4) encoded by SEX4 gene has recently been intensively studied because of its vital role in the degradation of leaf starch. In this study, we isolated and chromosomally mapped barley SEX4, characterized its gene and protein structure, predicted the cis-elements of its promoter, and analysed its expression based on real-time quantitative PCR and publically available microarray data. The full length of barely SEX4 (HvSEX4) was 4,598 bp and it was mapped on the long arm of chromosome 4H (4HL). This gene contained 14 exons and 13 introns in all but two of the species analysed, Arabidopsis (13 exons and 12 introns) and Oryza brachyantha (12 exons and 11 introns). An exon-intron junction composed of intron 4 to intron 7 and exon 5 to exon 8 was highly conserved among the analysed species. SEX4 is characterized with conserved functional domains (dual specificity phosphatase domain and carbohydrate-binding module 48) and varied chloroplast transit peptide and C-terminal. Expression analyses indicated that: (1) SEX4 was mainly expressed in anthers of barley, young leaf and anthers of rice, and leaf of Arabidopsis; (2) it exhibited a diurnal pattern in barley, rice and Arabidopsis; (3) significant difference in the expression of SEX4 was not detected for either barley or rice under any of the investigated stresses; and (4) it was significantly down-regulated at middle stage and up-regulated at late stage under cold treatment, down-regulated at early stage under heat treatment, and up-regulated at late stage under salt treatment in Arabidopsis. The strong relationships detected in the current study between SEX4 and glucan, water dikinases (GWD) or phosphoglucan, water dikinases (PWD) were discussed. Collectively, our results provide insights into genetic manipulation of SEX4, especially in monocotyledon and uncovering the possible roles of SEX4 in plant development.

ATP-binding cassette transporter TaABCG2 contributes to Fusarium head blight resistance by mediating salicylic acid transport in wheat.

ATP-binding cassette (ABC) transporters hydrolyse ATP to transport various substrates. Previous studies have shown that ABC transporters are responsible for transporting plant hormones and heavy metals, thus contributing to plant immunity. Herein, we identified a wheat G-type ABC transporter, TaABCG2-5B, that responds to salicylic acid (SA) treatment and is induced by Fusarium graminearum, the primary pathogen causing Fusarium head blight (FHB). The loss-of-function mutation of TaABCG2-5B (ΔTaabcg2-5B) reduced SA accumulation and increased susceptibility to F. graminearum. Conversely, overexpression of TaABCG2-5B (OE-TaABCG2-5B) exerted the opposite effect. Quantification of intracellular SA in ΔTaabcg2-5B and OE-TaABCG2-5B protoplasts revealed that TaABCG2-5B acts as an importer, facilitating the transport of SA into the cytoplasm. This role was further confirmed by Cd2+ absorption experiments in wheat roots, indicating that TaABCG2-5B also participates in Cd2+ transport. Thus, TaABCG2-5B acts as an importer and is crucial for transporting multiple substrates. Notably, the homologous gene TaABCG2-5A also facilitated Cd2+ uptake in wheat roots but did not significantly influence SA accumulation or FHB resistance. Therefore, TaABCG2 could be a valuable target for enhancing wheat tolerance to Cd2+ and improving FHB resistance.

Structure and expression of barley starch phosphorylase genes.

The function of starch phosphorylase has long been debated on the regulation of starch metabolism during the growth and development of plants. In this study, we isolated starch phosphorylase genes (Pho1 and Pho2) from barley, characterized their gene and protein structures, predicated their promoter's cis-elements and analyzed expression patterns. Multiple alignments of these genes showed that (1) both Pho1 and Pho2 genes possess 15 exons and 14 introns in all but three of the species analyzed, Aegilops tauschii (for Pho1 which contains 16 exons and 15 introns), potato (for Pho1b which contains 14 exons and 13 introns), and Triticum uraru (for Pho2 which contains 15 exons and 14 introns); (2) the exon-intron junctions of Pho1 and Pho2 flanking the ligand-binding sites are more conservative than the other regions. Analysis of protein sequences revealed that Pho1 and Pho2 were highly homologous except for two regions, the N terminal domain and the L78 insertion region. The results of real-time quantitative PCR (RT-qPCR) indicated that Pho2 is mainly expressed in germinating seeds, and the expression of Pho1 is similar to that of starch synthesis genes during seed development in barley. Microarray-based analysis indicated that the accumulation of Pho1 or Pho2 transcripts exhibited uniform pattern both in various tissues and various stages of seed development among species of barley, rice, and Arabidopsis. Pho1 of barley was significantly down-regulated under cold and drought treatments, and up-regulated under stem rust infection. Pho2 exhibited similar expression to Pho1 in barley. However, significant difference in expression was not detected for either Pho1 or Pho2 under any of the investigated abiotic stresses. In Arabidopsis, significant down-regulation was detected for Pho1 (PHS1) under abscisic acid (ABA) and for Pho2 (PHS2) under cold, salt, and ABA. Our results provide valuable information to genetically manipulate phosphorylase genes and to further elucidate their regulatory mechanism in the starch biosynthetic pathway.

Characterization and expression analysis of waxy alleles in barley accessions.

Granule Bound Starch Synthase I (GBSS I) encoded by the waxy gene plays an important role in accumulating amylose during the development of starch granules in barley. In this study, we isolated and characterized waxy alleles of three waxy (GSHO 908, GSHO 1828 and NA 40) and two non-waxy barley accessions (PI 483237 and CIho 15773), estimated the expression patterns of waxy genes via Real-time quantitative PCR (RT-qPCR), investigated promoter activity by analyzing promoter-GUS expression, and examined possible effects of waxy alleles on starch granule morphology in barley accessions by scanning electron microscopy (SEM). A 193-bp insertion in intron 1, a 15-bp insertion in the coding region, and some single nucleotide polymorphic sites were detected in the waxy barley accessions. In addition, a 397-bp deletion containing the TATA box, transcription starting point, exon 1 and partial intron 1 were also identified in the waxy barley accessions. RT-qPCR analysis showed that waxy accessions had lower waxy expression levels than those of non-waxy accessions. Transient expression assays showed that GUS activity driven by the 1,029-bp promoter of the non-waxy accessions was stronger than that driven by the 822-bp promoter of the waxy accessions. SEM revealed no apparent differences of starch granule morphology between waxy and non-waxy accessions. Our results showed that the 397-bp deletion identified in the waxy barley accessions is likely responsible for the reduction of waxy transcript, leading to lower concentrations of GBSS I protein thus lower amylose content.

Novel variants of HMW glutenin subunits from Aegilops section Sitopsis species in relation to evolution and wheat breeding.

BACKGROUND: High molecular weight glutenin subunits (HMW-GSs), encoded by the genes at Glu-1 loci in wheat and its related species, are significant in the determination of grain processing quality. However, the diversity and variations of HMW-GSs are relatively low in bread wheat. More interests are now focused on wheat wild relatives in Triticeae. The genus Aegilops represents an important germplasm for novel HWM-GSs and other useful genes for wheat genetic improvement. RESULTS: Six novel Glu-1 alleles and HMW-GSs were identified and characterized from three species of Aegilops section Sitopsis (S genome). Both open reading frames (ORFs) and promoter regions of these Glu-1 alleles were sequenced and characterized. The ORFs of Sitopsis Glu-1 genes are approximately 2.9 kb and 2.3 kb for x-type and y-type subunits, respectively. Although the primary structures of Sitopsis HMW-GSs are similar to those of previously reported ones, all six x-type or y-type subunits have the large fragment insertions. Our comparative analyses of the deduced amino acid sequences verified that Aegilops section Sitopsis species encode novel HMW-GSs with their molecular weights larger than almost all other known HMW-GSs. The Glu-1 promoter sequences share the high homology among S genome. Our phylogenetic analyses by both network and NJ tree indicated that there is a close phylogenetic evolutionary relationship of x-type and y-type subunit between S and D genome. CONCLUSIONS: The large molecular weight of HMW-GSs from S genome is a unique feature identified in this study. Such large subunits are resulted from the duplications of repetitive domains in Sitopsis HMW-GSs. The unequal crossover events are the most likely mechanism of variations in glutenin subunits. The S genome-encoded subunits, 1Dx2.2 and 1Dx2.2* have independent origins, although they share similar evolutionary mechanism. As HMW-GSs play a key role in wheat baking quality, these large Sitopsis glutenin subunits can be used as special genetic resources for wheat quality improvement.