Mei Deng

Genome-wide association study of phosphorus-deficiency-tolerance traits in Aegilops tauschii

Key messageUsing GWAS, 13 significant SNPs distributed on six of the sevenAegilops tauschiichromosomes (all but 5D) were identified, and several candidate P-deficiency-responsive genes were proposed from searches of public databases.AbstractAegilops tauschii, the wheat (Triticum aestivum) D-genome progenitor, possesses numerous genes for stress resistance, including genes for tolerance of phosphorus (P) deficiency. Investigation of the genetic architecture of A. tauschii will help in developing P-deficiency-tolerant varieties of wheat. We evaluated nine traits in a population of 380 A. tauschii specimens under conditions with and without P application, and we performed genome-wide association studies for these traits using single nucleotide polymorphism (SNP) chips containing 7185 markers. Using a general linear model, we identified 119 SNPs that were significantly associated with all nine traits, and a mixed linear model revealed 18 SNPs associated with all traits. Both models detected 13 significant markers distributed on six of the seven A. tauschii chromosomes (all but 5D). Searches of public databases revealed several candidate/flanking genes related to P-deficiency tolerance. These genes were grouped in five categories by the types of proteins they encoded: defense response proteins, enzymes, promoters and transcription factors, storage proteins, or proteins triggered by P deficiency. The identified SNPs and genes contain essential information for cloning genes related to P-deficiency tolerance in A. tauschii and wheat, and they provide a foundation for breeding P-deficiency tolerant wheat cultivars.

Identification of QTL for flag leaf length in common wheat and their pleiotropic effects

Leaf size is an important factor contributing to the photosynthetic capability of wheat plants. It also significantly affects various agronomic traits. In particular, the flag leaves contribute significantly to grain yield in wheat. A recombinant inbred line (RIL) population developed between varieties with significant differences in flag leaf traits was used to map quantitative trait loci (QTL) of flag leaf length (FLL) and to evaluate its pleiotropic effects on five yield-related traits, including spike length (SL), spikelet number per spike (SPN), kernel number per spike (KN), kernel length (KL), and thousand-kernel weight (TKW). Two additional RIL populations were used to validate the detected QTL and reveal the relationships in different genetic backgrounds. Using the diversity arrays technology (DArT) genetic linkage map, three major QTL for FLL were detected, with single QTL in different environments explaining 8.6–23.3% of the phenotypic variation. All the QTL were detected in at least four environments, and validated in two related populations based on the designed primers. These QTL and the newly developed primers are expected to be valuable for fine mapping and marker-assisted selection in wheat breeding programs.

Linoleic acid isomerase gene FgLAI12 affects sensitivity to salicylic acid, mycelial growth and virulence of Fusarium graminearum

Fusarium graminearum is the major causal agent of fusarium head blight in wheat, a serious disease worldwide. Linoleic acid isomerase (LAI) catalyses the transformation of linoleic acid (LA) to conjugated linoleic acid (CLA), which is beneficial for human health. We characterised a cis-12 LAI gene of F. graminearum (FGSG_02668; FgLAI12), which was downregulated by salicylic acid (SA), a plant defence hormone. Disruption of FgLAI12 in F. graminearum resulted in decreased accumulation of cis-9,trans-11 CLA, enhanced sensitivity to SA, and increased accumulation of LA and SA in wheat spikes during infection. In addition, mycelial growth, accumulation of deoxynivalenol, and pathogenicity in wheat spikes were reduced. Re-introduction of a functional FgLAI12 gene into ΔFgLAI12 recovered the wild-type phenotype. Fluorescent microscopic analysis showed that FgLAI12 protein was usually expressed in the septa zone of conidia and the vacuole of hyphae, but was expressed in the cell membrane of hyphae in response to exogenous LA, which may be an element of LA metabolism during infection by F. graminearum. The cis-12 LAI enzyme encoded by FgLAI12 is critical for fungal response to SA, mycelial growth and virulence in wheat. The gene FgLAI12 is potentially valuable for biotechnological synthesis of cis-9,trans-11 CLA.

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.

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.

Genetic analyses of Glu-1S sh in wheat/Aegilops sharonensis hybrid progenies and development of alien HMW-GSs gene-specific markers

Aegilops sharonensis is a diploid relative of wheat and has novel high molecular weight glutenin subunits (HMW-GSs) encoded by Glu-1Ssh. Hybrids of tetraploid wheat species and Ae. sharonensis have been successfully created by wild crosses. In our study, we conducted backcrosses and introduced the novel HMW-GSs of Ae. sharonensis into hexaploid wheat species. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and cytogenetics indicated that lines carrying the HMW-GSs encoded by Glu-1Ssh had varied chromosome numbers ranging from 35 to 50. Among these, three lines with 42 chromosomes (25-8, 33-1, and 42-2) were identified by visual examination. Except for possessing the alien HMW-GSs of Ae. sharonensis, the agricultural traits of the three lines were similar to those of common wheat, indicating their potential for wheat breeding. Additionally, we developed two molecular markers specific to the x- and y-type HMW-GSs genes of Ae. sharonensis. These two molecular markers were validated in backcross progenies and were proven to accurately trace and distinguish Ae. sharonensisGlu-1Ssh from Glu-1A, Glu-1B, and Glu-1D of wheat. Therefore, the Glu-1Ssh HMW-GSs lines with 42 chromosomes, together with gene-specific markers, will be valuable for molecular-assisted selection in alien genes utilization and wheat breeding.

Conferring resistance to pre-harvest sprouting in durum wheat by a QTL identified in Triticum spelta

Pre-harvest sprouting (PHS) causes significant yield loss and degrade the end-use quality of wheat, especially in regions with prolonged wet weather during the harvesting season. Unfortunately, the gene pool of Triticum durum (tetraploid durum wheat) has narrow genetic base for PHS resistance. Therefore, finding out new genetic resources from other wheat species to develop PHS resistance in durum wheat is of importance. A major PHS resistance QTL, Qphs.sicau-3B.1, was mapped on chromosome 3BL in a recombinant inbred line population derived from ‘CSCR6’ (Triticum spelta), a PHS resistant hexaploid wheat and ‘Lang’, a PHS susceptible Australian hexaploid wheat cultivar. This QTL, Qphs.sicau-3B.1, is positioned between DArT marker wPt-3107 and wPt-6785. Two SCAR markers (Ph3B.1 and Ph3B.2) were developed to track this major QTL and were used to assay a BC2F8 tetraploid population derived from a cross between the durum wheat ‘Bellaroi’ (PHS susceptible) and ‘CSCR6’ (PHS resistant). Phenotypic assay and marker-assisted selection revealed five stable tetraploid lines were highly PHS resistant. This study has successfully established that PHS-resistance QTL from hexaploid wheat could be efficiently introgressed into tetraploid durum wheat. This tetraploid wheat germplasm could be useful in developing PHS resistant durum cultivars with higher yield and good end-use quality.

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.

Variation and diversity of the breakpoint sequences on 4AL for the 4AL/5AL translocation in Triticum

The translocation of 4AL/5AL in Triticum, which occurred before the differentiation of T. urartu and einkorn, is an important chromosomal rearrangement. Recently, the first identification of breakpoint sequence on 4AL for this translocation provides the opportunity to analyze the variation and diversity of breakpoints in Triticum. In this study, the breakpoint regions of 52 accessions from 21 species were isolated and further characterized. The sequences were divided into 12 types based on their lengths, which ranged from 2009 to 2552 bp. Cluster analysis showed that they were further divided into three groups. Interesting evolutionary relationships among a few of the species were observed and discussed. Multiple sequence alignment of the 52 sequences made it possible to detect 13 insertion and deletion length polymorphisms (InDels) and 101 single nucleotide polymorphisms (SNPs). Furthermore, several species- or accession-specific SNPs or InDels were also identified. Based on BLAST analysis of the conserved sequences, the breakpoint was narrowed down to a 125 bp fragment. Taken together, the results obtained in this study enrich our understanding of chromosomal breakpoints and will be useful for the identification of other breakpoints in wheat.

Cloning and characterization of Agp1, the gene encoding the small subunit of ADP-glucose pyrophosphorylase from wheat and its relatives

Abstract ADP-glucose pyrophosphorylase (AGP) consists of two large (AGP-L) and two small (AGP-S) subunits; it regulates the limiting step in the biosynthesis of starch. Here, we isolated the full-length cDNA sequence of the AGP-S gene (designed as Agp1) from wheat and whole genomic sequences from relatives of wheat. The coding region of Agp1 ranged from 5948 bp to 7666 bp and contained 9 exons and 8 introns. The full-length cDNA sequence of Agp1 encoded a polypeptide containing 473 amino acids without transit peptides. The sequence alignment and phylogenetic tree analyses indicated that the Agp1 cDNA sequence was highly conserved (99.7%) across the diploid Triticeae species examined in this study. Variations in introns caused the Agp1 genomic sequences from wheat relatives to be less similar (79.5%), and Agp1 sequences from the B genome donor species were divergent. In developing grains, rapid increase in Agp1 expression at prophase was closely followed by the accumulation of starch, and a significant (P < 0.001) correlation was observed between them. The heterogeneous mixtures of small and large subunits from different wheat species restarted glycogen synthesis in the Escherichia coli AC70R1-504 cells, which are deficient in endogenous AGP activity (glg C−/−). Our data provide useful information to evaluate and utilize Agp1 sequences in Triticeae species.