Shouzhong Zhang

Development and molecular cytogenetic analysis of wheat-Haynaldia villosa 6VS/6AL translocation lines specifying resistance to powdery mildew

Several Triticum aestivum L.-Haynaldia villosa disomic 6VS/6AL translocation lines with powdery mildew resistance were developed from the hybridization between common wheat cultivar Yangmai 5 and alien substitution line 6V(6A). Mitotic and meiotic C-banding analysis, aneuploid analysis with double ditelosomic stocks, in situ hybridization, as well as the phenotypic assessment of powdery mildew resistance, were used to characterize these lines. The same translocated chromosome, with breakpoints near the centromere, appears to be present in all the lines, despite variation among the lines in their morphology and agronomic characteristics. The resistance gene, conferred by H. villosa and designated as Pm21, is a new and promising source of powdery mildew resistance in wheat breeding.

QTLs for fusarium head blight response in a wheat DH population of Wangshuibai/Alondra's'

SummaryA doubled haploid (DH) wheat population derived from the cross Wangshuibai/Alondra‘s’ was developed through chromosome doubling of haploids generated by anther culture of hybrids. Fusarium head blight (FHB) was evaluated for three years from 2001 to 2003 in Jianyang, Fujian Province, China, where epidemics of FHB have been consistently severe. After 307 pairs of simple sequence repeat (SSR) primers were screened, 110 pairs were polymorphic between Wangshuibai and Alondra`s’, and used to construct a genetic linkage map for detection of quantitative trait loci (QTLs). A stable QTL for low FHB severity was detected on chromosomes 3B over all three years, and QTLs on chromosomes 5B, 2D, and 7A were detected over two years. Additional QTLs on chromosomes 3A, 3D, 4B, 5A, 5D, 6B and 7B showed marginal significance in only one year. Six QTLs were detected when phenotypic data from three years were combined. In addition, significant additive-by-additive epistasis was detected for a QTL on 6A although its additive effect was not significant. Additive effects (A) and additive-by-additive epistasis (AA) explained a major portion of the phenotypic variation (76.5%) for FHB response. Xgwm533-3B and Xgwm335-5B were the closest markers to QTLs, and have potential to be used as selectable markers for marker-assisted selection (MAS) in wheat breeding programs.

Development and characterization of wheat- Leymus racemosus translocation lines with resistance to Fusarium Head Blight

Wheat scab (Fusarium Head Blight, FHB) is a destructive disease in the warm and humid wheat-growing areas of the world. Finding diverse sources of FHB resistance is critical for genetic diversity of resistance for wheat breeding programs. Leymus racemosus is a wild perennial relative of wheat and is highly resistant to FHB. Three wheat- L. racemosus disomic addition (DA) lines DA5Lr#1, DA7Lr#1 and DALr.7 resistant to FHB were used to develop wheat- L.racemosus translocation lines through irradiation and gametocidal gene-induced chromosome breakage. A total of nine wheat-alien translocation lines with wheat scab resistance were identified by chromosome C-banding, GISH, telosomic pairing and RFLP analyses. In line NAU614, the long arm of 5Lr#1 was translocated to wheat chromosome 6B. Four lines, NAU601, NAU615, NAU617, and NAU635, had a part of the short arm of 7Lr#1 transferred to different wheat chromosomes. Four other lines, NAU611, NAU634, NAU633, and NAU618, contained translocations involving Leymus chromosome Lr.7 and different wheat chromosomes. The resistance level of the translocation lines with a single alien chromosome segment was higher than the susceptible wheat parent Chinese Spring but lower than the alien resistant parent L. racemosus. At least three resistance genes in L. racemosus were identified. One was located on chromosome Lr.7, and two could be assigned to the long arm of 5Lr#1 and the short arm of 7Lr#1.

Effects of the 6VS.6AL translocation on agronomic traits and dough properties of wheat

Nineteen common wheat cultivars and advanced lines carrying a 6VS.6AL translocation and five parents were sown at two locations in Jiangsu in 2004–05 season to assess the effects of the translocation on grain yield and dough properties. In general, there were no significant differences between 6VS.6AL lines and their recurrent parents in agronomic, mixograph and starch pasting traits, including grain yield, grains/spike, grain weight/spike, mixing time and peak viscosity. 6VS.6AL lines showed slightly but significantly higher thousand-kernel weight and plant height, and small negative effects on test weight, flour yield and flour colour. However, significant variation occurred for all traits among sister lines from the same cross, indicating that additional selection could lead to further improvement. It was concluded that the 6VS.6AL translocation can be used in wheat breeding programs as a donor of resistance to powdery mildew with no obvious undesirable effects on agronomic and quality traits.

Two members of TaRLK family confer powdery mildew resistance in common wheat

BackgroundPowdery mildew, caused by Blumeria graminearum f.sp. tritici (Bgt), is one of the most severe fungal diseases of wheat. The exploration and utilization of new gene resources is the most effective approach for the powdery mildew control.ResultsWe report the cloning and functional analysis of two wheat LRR-RLKs from T. aestivum c.v. Prins- T. timopheevii introgression line IGV1-465, named TaRLK1 and TaRLK2, which play positive roles in regulating powdery mildew resistance in wheat. The two LRR-RLKs contain an ORF of 3,045 nucleotides, encoding a peptide of 1014 amino acids, with seven amino acids difference. Their predicted proteins possess a signal peptide, several LRRs, a trans-membrane domain, and a Ser/Thr protein kinase domain. In response to Bgt infection, the TaRLK1/2 expression is up-regulated in a developmental-stage-dependent manner. Single-cell transient over-expression and gene-silencing assays indicate that both genes positively regulate the resistance to mixed Bgt inoculums. Transgenic lines over-expressing TaRLK1 or TaRLK2 in a moderate powdery mildew susceptible wheat variety Yangmai 158 led to significantly enhanced powdery mildew resistance. Exogenous applied salicylic acid (SA) or hydrogen peroxide (H2O2) induced the expression of both genes, and H2O2 had a higher accumulation at the Bgt penetration sites in RLK over-expression transgenic plants, suggesting a possible involvement of SA and altered ROS homeostasis in the defense response to Bgt infection. The two LRR-RLKs are located in the long arm of wheat chromosome 2B, in which the powdery mildew resistance gene Pm6 is located, but in different regions.ConclusionsTwo members of TaRLK family were cloned from IGV1-465. TaRLK1 and TaRLK2 contribute to powdery mildew resistance of wheat, providing new resistance gene resources for wheat breeding.

Development and characterization of a complete set of Triticum aestivum–Roegneria ciliaris disomic addition lines

Key messageA complete set wheat-R. ciliaris disomic addition lines (DALs) were characterized and the homoeologous groups and genome affinities of R. ciliaris chromosomes were determined.AbstractWild relatives are rich gene resources for cultivated wheat. The development of alien addition chromosome lines not only greatly broadens the genetic diversity, but also provides genetic stocks for comparative genomics studies. Roegneria ciliaris (genome ScScYcYc), a tetraploid wild relative of wheat, is tolerant or resistant to many abiotic and biotic stresses. To develop a complete set of wheat-R. ciliaris disomic addition lines (DALs), we undertook a euplasmic backcrossing program to overcome allocytoplasmic effects and preferential chromosome transmission. To improve the efficiency of identifying chromosomes from Sc and Yc, we established techniques including sequential genomic in situ hybridization/fluorescence in situ hybridization (FISH) and molecular marker analysis. Fourteen DALs of wheat, each containing one pair of R. ciliaris chromosomes pairs, were characterized by FISH using four repetitive sequences [pTa794, pTa71, RcAfa and (GAA)10] as probes. One hundred and sixty-two R. ciliaris-specific markers were developed. FISH and marker analysis enabled us to assign the homoeologous groups and genome affinities of R. ciliaris chromosomes. FHB resistance evaluation in successive five growth seasons showed that the amphiploid, DA2Yc, DA5Yc and DA6Sc had improved FHB resistance, indicating their potential value in wheat improvement. The 14 DALs are likely new gene resources and will be phenotyped for more agronomic performances traits.

Whole genome development of intron targeting (IT) markers specific for Dasypyrum villosum chromosomes based on next-generation sequencing technology

Dasypyrum villosum (Dv), a wild relative of wheat, is an important and useful gene resource for wheat improvement. A large number of wheat-Dv aneuploid lines harboring whole or fragments of Dv chromosomes have been developed. However, the lack of sufficient molecular markers hindered accurate identification of Dv chromatin, especially when the introgressed fragments are small. Development of molecular markers covering the whole Dv genome and evenly distributed on different chromosome regions is not only useful for the detection of the introgressed alien chromatin in wheat background, but also provides evidence of the syntenic relationship between homoeologous chromosomes. In the present study, in order to develop high density and evenly distributed molecular markers on individual Dv chromosomes, genomic DNA of Dv leaves was sequenced and assembled. Sequence assemblies of all wheat chromosomes were first used to identify exon–exon junctions and localize introns in Dv. Intron length polymorphisms suitable for designing Dv primers flanking introns were evaluated, and a total of 1624 intron targeting (IT) markers was designed. By using the Chinese Spring, the Triticum durum-Dv amphiploid and the Dv sequenced DNA libraries, 841 IT molecular markers specific for Dv chromosomes were developed, with maximum efficiency up to 51.79%. We assigned the 841 IT markers to seven Dv chromosomes (1V–7V) using seven wheat–Dv chromosome addition and substitution lines: 135 to 1V, 175 to 2V, 120 to 3V, 89 to 4V, 140 to 5V, 71 to 6V, and 111 to 7V, respectively. Using T. aestivum-Dv telosomic and whole arm translocation lines, they were further located on the short or long chromosome arms. These specific markers for individual chromosomes of Dv provided efficient tools for the characterization of structural variation involving the individual chromosome of Dv, as well as for the selection of useful genes located on individual Dv chromosome in breeding programs.

TaNAC6s are involved in the basal and broad-spectrum resistance to powdery mildew in wheat

NACs are important transcriptional factors involved in growth and development as well as responses to abiotic and biotic stresses in plants. In this study, TaNAC6 was identified as a differentially expressed gene between two lines with broad-spectrum resistance to powdery mildew, NAU9918 and OEStpk-V, and their corresponding susceptible isogenic lines, SM-1 and Yangmai158, after Bgt inoculation by transcriptome analysis. Then, three homoeologous genes of TaNAC6 were cloned and named as TaNAC6-A, TaNAC6-B and TaNAC6-D, respectively. Each member of TaNAC6s was subcellular localized to the nucleus and displayed the transcriptional activation activity. However, the responses of them to pathogens and phytohormones were different. Transient overexpression of each TaNAC6 reduced the haustorium index of Yangmai158, and stable transformation of TaNAC6-A enhanced its resistance against Bgt, implying that TaNAC6s play important roles in basal resistance. Silencing of TaNAC6s compromised the resistance of OEStpk-V and NAU9918 suggesting that TaNAC6s play positive roles in the broad-spectrum resistance against Bgt. TaNAC6s might be induced by JA and then feedback regulate the JA pathway leading to improved resistance to Bgt. The role of TaNAC6s and their orthologous genes HvNAC6 and ATAF1 in the powdery mildew resistance implied these NAC6 genes share a common signal pathway across species.

A malectin-like/leucine-rich repeat receptor protein kinase gene, RLK-V, regulates powdery mildew resistance in wheat: A malectin-like/LRR receptor protein kinase gene

Pattern recognition receptors (PRRs) can trigger plant immunity through the recognition of pathogen-associated molecular patterns. In this study, we report that a malectin-like/leucine-rich repeat receptor protein kinase gene, RLK-V, from Haynaldia villosa putatively acts as a PRR to positively regulate powdery mildew resistance caused by Blumeria graminis f. sp. tritici (Bgt) in wheat. RLK-V has two alternatively spliced transcripts corresponding to an intact RLK-V1.1 and a truncated RLK-V1.2 caused by intron retention. Expression analysis showed that both transcripts could be up-regulated by Bgt in resistant materials, whereas the functional RLK-V1.1 was expressed only after Bgt inoculation. Promoter activity assays indicated that RLK-V could respond to Bgt even in susceptible wheat. Silencing of RLK-V in Pm21-carrying resistant materials resulted in compromised resistance to Bgt. In addition, over-expression of RLK-V1.1 in Pm21-lacking susceptible Yangmai158 and SM-1 by single-cell transient expression and stable transformation in Yangmai158 could improve powdery mildew resistance. We propose that RLK-V regulates basal resistance to powdery mildew, which is also required for broad-spectrum resistance mediated by the Pm21 gene. Over-expression of RLK-V1.1 could trigger cell death in Nicotiana benthamiana, and RLK-V1.1 transgenic wheat accumulated more reactive oxygen species and displayed a stronger hypersensitive response than did the recipient, which led to enhanced Bgt resistance. However, constitutive activation of RLK-V1.1 resulted in the abnormal growth of transgenic plants.