Genome-wide quantitative trait locus mapping identifies multiple major loci for brittle rachis and threshability in Tibetan semi-wild wheat (Triticum aestivum ssp. tibetanum Shao).

Tibetan semi-wild wheat (Triticum aestivum ssp. tibetanum Shao) is a semi-wild hexaploid wheat resource that is only naturally distributed in the Qinghai-Tibet Plateau. Brittle rachis and hard threshing are two important characters of Tibetan semi-wild wheat. A whole-genome linkage map of T. aestivum ssp. tibetanum was constructed using a recombinant inbred line population (Q1028×ZM9023) with 186 lines, 564 diversity array technology markers, and 117 simple sequence repeat markers. Phenotypic data on brittle rachis and threshability, as two quantitative traits, were evaluated on the basis of the number of average spike rachis fragments per spike and percent threshability in 2012 and 2013, respectively. Quantitative trait locus (QTL) mapping performed using inclusive composite interval mapping analysis clearly identified four QTLs for brittle rachis and three QTLs for threshability. However, three loci on 2DS, 2DL, and 5AL showed pleiotropism for brittle rachis and threshability; they respectively explained 5.3%, 18.6%, and 18.6% of phenotypic variation for brittle rachis and 17.4%, 13.2%, and 35.2% of phenotypic variation for threshability. A locus on 3DS showed an independent effect on brittle rachis, which explained 38.7% of the phenotypic variation. The loci on 2DS and 3DS probably represented the effect of Tg and Br1, respectively. The locus on 5AL was in very close proximity to the Q gene, but was different from the predicted q in Tibetan semi-wild wheat. To our knowledge, the locus on 2DL has never been reported in common wheat but was prominent in T. aestivum ssp. tibetanum accession Q1028. It remarkably interacted with the locus on 5AL to affect brittle rachis. Several major loci for brittle rachis and threshability were identified in Tibetan semi-wild wheat, improving the understanding of these two characters and suggesting the occurrence of special evolution in Tibetan semi-wild wheat.

The γ-gliadin-like γ-prolamin genes in the tribe Triticeae.

The γ-prolamins are important components of seed storage proteins in wheat and other Triticeae species. Here, the γ-prolamin genes from the diploid Triticeae species were systemically characterized. Most of the γ-prolamins (except 75 K γ-secalins) characterized were defined as γ-gliadin-like γ-prolamins, since they shared same characteristic model structure with γ-gliadins. Over one-third of these putatively functional γ-prolamin peptides contained different number of cysteine residues as compared to the eight residues present in γ-gliadins. Sequence polymorphism and linkage disequilibrium analyses showed the conservation of γ-prolamin genes in Triticeae species under evolutionary selection. Phylogenetic analyses indicated that these γ-prolamin genes can not be clearly separated according to their genomic origins, reflecting the conservation of γ-gliadinlike γ-prolamin genes after the divergence of Triticeae species. A screening of coeliac disease (CD) toxic epitopes shows that the γ-prolamins from some other genomes contain much fewer epitopes than those from the A, S (B) and D genomes of wheat. These findings contribute to better understanding of γ-prolamin family in Triticeae and build a ground for breeding less CD-toxic wheat cultivars.

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.

Characterization of HMW-GSs and their gene inaction in tetraploid wheat.

In this study, we report the expression of HMW-GSs in 87 accessions of tetraploid wheat, the characterization of three inactive and one active HMW glutenin genes, and the functional verification of HMW-GSs by promoter-GUS expression. SDS-PAGE profiles revealed that tetraploid wheat has many different combinations of HMW-GSs and the number of subunits varies from 1 to 4. HMW glutenin genes at the Glu-A1x, Glu-A1y and Glu-B1y loci exhibited different frequencies of inaction while the Glu-B1x allele was expressed in all 87 accessions. Gene cloning showed that only 1Bx (Tdu-e) could express a full-length protein and its deduced protein sequence has the typical primary structure but with fewer cysteine residues. The expression of the other three HMW glutenin genes has been disrupted by stop codons in their repetitive domains. Besides short indels or mutations of one or more bases, an 85-bp deletion and a 185-bp insertion were found in the promoter regions of 1Ay (Tdu-s) and 1Bx (Tdu-e). The transient expression of promoter-GUS constructs indicated that the 1Ay promoter can drive expression of the GUS gene. We conclude that defects (stop codons or the insertion of large transposon-like elements) in the coding regions may be the most probable cause for the inaction of the HMW glutenin genes.

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.

Characterization of barley Prp1 gene and its expression during seed development and under abiotic stress.

The pre-mRNA processing (Prp1) gene encodes a spliceosomal protein. It was firstly identified in fission yeast and plays a regular role during spliceosome activation and cell cycle. Plant Prp1 genes have only been identified from rice, Sorghum and Arabidopsis thaliana. In this study, we reported the identification and isolation of a novel Prp1 gene from barley, and further explored its expressional pattern by using real-time quantitative RTPCR, promoter prediction and analysis of microarray data. The putative barley Prp1 protein has a similar primary structure features to those of other known Prp1 protein in this family. The results of amino acid comparison indicated that Prp1 protein of barley and other plant species has a highly conserved 30 termnal region while their 50 sequences greatly varied. The results of expressional analysis revealed that the expression level of barley Prp1 gene is always stable in different vegetative tissues, except it is up-regulated at the mid- and late stages of seed development or under the condition of cold stress. This kind of expressional pattern for barley Prp1 is also supported by our results of comparison of microarray data from barley, rice and Arabidopsis. For the molecular mechanism of its expressional pattern, we conclude that the expression of Prp1 gene may be up-regulated by the increase of pre-mRNAs and not be constitutive or ubiquitous.

Phylogenetic inferences in Avena based on analysis of FL intron2 sequences.

The development and application of molecular methods in oats has been relatively slow compared with other crops. Results from the previous analyses have left many questions concerning species evolutionary relationships unanswered, especially regarding the origins of the B and D genomes, which are only known to be present in polyploid oat species. To investigate the species and genome relationships in genus Avena, among 13 diploid (A and C genomes), we used the second intron of the nuclear gene FLORICAULA/LEAFY (FL int2) in seven tetraploid (AB and AC genomes), and five hexaploid (ACD genome) species. The Avena FL int2 is rather long, and high levels of variation in length and sequence composition were found. Evidence for more than one copy of the FL int2 sequence was obtained for both the A and C genome groups, and the degree of divergence of the A genome copies was greater than that observed within the C genome sequences. Phylogenetic analysis of the FL int2 sequences resulted in topologies that contained four major groups; these groups reemphasize the major genomic divergence between the A and C genomes, and the close relationship among the A, B, and D genomes. However, the D genome in hexaploids more likely originated from a C genome diploid rather than the generally believed A genome, and the C genome diploid A. clauda may have played an important role in the origination of both the C and D genome in polyploids.

[Isolation and characterization of a low molecular weight glutenin gene from Taenitherum Nevski].

More and more low-molecular-weight glutenin(LMW glutenin) genes were isolated and characterized from hexaploid wheat (Triticum aestivum L.). However, few homologous genes were obtained from its relative species, which limited our understanding of the relationships among them. Therefore, it is necessary to isolate LMW glutenin homologous genes from wheat wild relative species. Using a pair of specific oligonucleotide PCR primers for Taenitherum genomic DNA, a LMW glutenin gene sequence, with nucleotide sequence in 1 035 bp and deduced amino acid sequence with 343 amino acid residues, was obtained. This sequence was a typical LMW glutenin sequence and characterized by a signal peptide of 21 amino acid residues, a N-terminal conservative domain of 13 amino acid residues, a repetitive domain of short peptide, and a C-terminal conservative domain. Sequence alignment showed the main differences and the relationships between LMW glutenin genes from wheat and Taenitherum. The results presented here give a reference to isolate LMW glutenin gene from Taenitherum, as well as other wheat wild relatives.

Characterization of two HMW glutenin subunit genes from Taenitherum Nevski.

The compositions of high molecular weight (HMW) glutenin subunits from three species of Taenitherum Nevski (TaTa, 2n = 2x = 14), Ta. caput-medusae, Ta. crinitum and Ta. asperum, were investigated by SDS-PAGE analysis. The electrophoresis mobility of the x-type HMW glutenin subunits were slower or equal to that of wheat HMW glutenin subunit Dx2, and the electrophoresis mobility of the y-type subunits were faster than that of wheat HMW glutenin subunit Dy12. Two HMW glutenin genes, designated as Tax and Tay, were isolated from Ta. crinitum, and their complete nucleotide coding sequences were determined. Sequencing and multiple sequences alignment suggested that the HMW glutenin subunits derived from Ta. crinitum had the similar structures to the HMW glutenin subunits from wheat and related species with a signal peptide, and N- and C-conservative domains flanking by a repetitive domain consisted of the repeated short peptide motifs. However, the encoding sequences of Tax and Tay had some novel modification compared with the HMW glutenin genes reported so far: (1) A short peptide with the consensus sequences of KGGSFYP, which was observed in the N-terminal of all known HMW glutenin genes, was absent in Tax; (2) There is a specified short peptide tandem of tripeptide, hexapeptide and nonapeptide and three tandem of tripeptide in the repetitive domain of Tax; (3) The amino acid residues number is 105 (an extra Q presented) but not 104 in the N-terminal of Tay, which was similar to most of y-type HMW glutenin genes from Elytrigia elongata and Crithopsis delileana. Phylogenetic analysis indicated that Tax subunit was mostly related to Ax1, Cx, Ux and Dx5, and Tay was more related to Ay, Cy and Ry.