Xiu-Jin Lan

Confirmation of the relationship between plant height and Fusarium head blight resistance in wheat (Triticum aestivum L.) by QTL meta-analysis

Fusarium head blight (FHB) is a serious wheat disease all over the world. In this study, the relationships between plant height (PH) and FHB were investigated across the whole wheat genome by QTL meta-analysis from fifty-six experiments. Coincident meta-QTL (MQTL) for PH and FHB were found on chromosomes 2D, 3A, 4B, 4D and 7A. Rht-B1, Rht-D1, Rht8, MQTLs P7 and P26 were consistent with FHB MQTLs. The meta-analysis results confirmed the negative associations of Rht-B1, Rht-D1, and Rht8 with FHB resistance. The associations of PH and FHB resistance on chromosomes 3A and 7A have not been reported and need further investigation. These regions should be given attention in breeding programs. MQTLs derived from several resistance sources were also observed. Some FHB MQTLs for different types of resistance overlapped. These findings could be useful for improving wheat varieties with resistance to FHB.

Rapid changes of microsatellite flanking sequence in the allopolyploidization of new synthesized hexaploid wheat

It was suggested that the rapid changes of DNA sequence and gene expression occurred at the early stages of allopolyploid formation. In this study, we revealed the microsatellite (SSR) differences between newly formed allopolyploids and their donor parents by using 21 primer sets specific for D genome of wheat. It was indicated that rapid changes had occurred in the “shock” process of the allopolyploid formation between tetraploid wheat and Aegilops tauschii. The changes of SSR flanking sequence resulted in appearance of novel bands or disappearance of parental bands. The disappearance of the parental bands showed much higher frequencies in comparison with that of appearance of novel bands. Disappearance of the parental bands was not random. The frequency of disappearance in tetraploid wheat was much higher than in Ae. tauschii, i. e. the disappearance frequency in AABB genome was much higher than in D genome. Changes of SSR flanking sequence occurred at the early stage of F1 hybrid or just after chromosome doubling. From the above results, it can be inferred that SSR flanking sequence region was very active and was amenable to change in the process of polyploidization. This suggested that SSR flanking sequence probably had special biological function at the early stage of ployploidization. The rapid and directional changes at the early stage of polyploidization might contribute to the rapid evolution of the newly formed allopolyploid and allow the divergent genomes to act in harmony.

TaCPK2-A, a calcium-dependent protein kinase gene that is required for wheat powdery mildew resistance enhances bacterial blight resistance in transgenic rice

Calcium-dependent protein kinases (CPKs) are important Ca2+ signalling components involved in complex immune and stress signalling networks; but the knowledge of CPK gene functions in the hexaploid wheat is limited. Previously, TaCPK2 was shown to be inducible by powdery mildew (Blumeria graminis tritici, Bgt) infection in wheat. Here, its functions in disease resistance are characterized further. This study shows the presence of defence-response and cold-response cis-elements on the promoters of the A subgenome homoeologue (TaCPK2-A) and D subgenome homoeologue (TaCPK2-D), respectively. Their expression patterns were then confirmed by quantitative real-time PCR (qRT-PCR) using genome-specific primers, where TaCPK2-A was induced by Bgt treatment while TaCPK2-D mainly responded to cold treatment. Downregulation of TaCPK2-A by virus-induced gene silencing (VIGS) causes loss of resistance to Bgt in resistant wheat lines, indicating that TaCPK2-A is required for powdery mildew resistance. Furthermore, overexpression of TaCPK2-A in rice enhanced bacterial blight (Xanthomonas oryzae pv. oryzae, Xoo) resistance. qRT-PCR analysis showed that overexpression of TaCPK2-A in rice promoted the expression of OsWRKY45-1, a transcription factor involved in both fungal and bacterial resistance by regulating jasmonic acid and salicylic acid signalling genes. The opposite effect was found in wheat TaCPK2-A VIGS plants, where the homologue of OsWRKY45-1 was significantly repressed. These data suggest that modulation of WRKY45-1 and associated defence-response genes by CPK2 genes may be the common mechanism for multiple disease resistance in grass species, which may have undergone subfunctionalization in promoters before the formation of hexaploid wheat.

HMW-glutenin and gliadin variations in Tibetan weedrace, Xinjiang rice wheat and Yunnan hulled wheat

Nine Tibetan weedrace, 9 Xingjiang rice wheat and 14 Yunnan hulled wheat accessions were evaluated for the variability of HMW-glutenins and gliadins. Higher variability was observed for both HMW-glutenins and gliadins in Tibetan weedrace and Xingjiang rice wheat, while lower variability was observed in Yunan hulled wheat. There were 4 HMW-glutenin and 9 gliadin patterns in 9 Tibetan weedrace accessions, 5 HMW-glutenin and 8 gliadin patterns in 9 Xingjiang rice wheat accessions, and 3 HMW-glutenin and 8 gliadin patterns in 14 Yunnan hulled wheat accessions. In Xinjiang rice wheat, one accession (i.e. Daomai 2) carried subunits 2.1 + 10.1 encoded by Glu-D1, which is very rare in common wheat.

QTL Mapping for Important Agronomic Traits in Synthetic Hexaploid Wheat Derived from Aegiliops tauschii ssp. tauschii

Aegiliops tauschii is classified into two subspecies: Ae. tauschii ssp. tauschii and Ae. tauschii ssp. strangulata. Novel genetic variations exist in Ae. tauschii ssp. tauschii that can be utilized in wheat improvement. We synthesized a hexaploid wheat genotype (SHW-L1) by crossing an Ae. tauschii ssp. tauschii accession (AS60) with a tetraploid wheat genotype (AS2255). A population consisting of 171 F8 recombinant inbred lines was developed from SHW-L1 and Chuanmai 32 to identify QTLs associated with agronomic traits. A new genetic map with high density was constructed and used to detect the QTLs for heading date, kernel width, spike length, spikelet number, and thousand kernel weight. A total of 30 putative QTLs were identified for five investigated traits. Thirteen QTLs were located on D genomes of SHW-L1, six of them showed positive effect on agronomic traits. Chromosome region flanked by wPt-6133–wPt-8134 on 2D carried five environment-independent QTLs. Each QTL accounted for more than 10% phenotypic variance. These QTLs were highly consistent across environments and should be used in wheat breeding.

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

BackgroundHigh 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.ResultsSix 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.ConclusionsThe 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.

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.

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.

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 Arabidopsisthaliana. 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 RT-PCR, 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 3′ termnal region while their 5′ 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.