Molecular markers and cytogenetics to characterize a wheat-Dasypyrum villosum 3V (3D) substitution line conferring resistance to stripe rust

Dasypyrum villosum has been used as a valuable gene resource for disease resistances, yield increase and quality improvement in wheat. A novel wheat-D. villosum alien introgression line CD-3 was generated through hybridization between the common wheat Chinese Spring (CS) and a CS-D. villosum 3V addition line having considerably high stripe rust resistance, which enable the characterization of a potential new stripe rust resistance gene (s) derived from D. villosum. The results of non-denaturing fluorescent in situ hybridization (ND-FISH) showed that CD-3 contained 42 chromosomes, including a 3V chromosome pair, and the absence of both of the 3D chromosomes. PCR-based Landmark Unique Gene (PLUG) molecular marker analysis supported results from the FISH analysis, revealing CD-3 was a wheat-D. villosum 3V (3D) disomic substitution line. Resistant test of stripe rust on 52 plants of F2 generation (CD-3/CS), CD-3, CS and D. villosum have been conducted at seedling stage. 7 plants of F2 generation possessing two 3V chromosomes exhibited high resistance to stripe rust as CD-3 and D. villosum, 10 plants carrying one 3V chromosome and 35 plants without 3V chromosome were susceptive to stripe rust as CS. The result implied the high stripe rust resistance of CD-3 should be controlled by recessive gene(s) originating from D. villosum. To rapidly detect chromosome 3V in the genetic background of wheat, we developed a novel Sequence Characterized Amplified Region (SCAR) marker specific for 3V chromosome based on the sequence of a grain size-related gene DvGS5 in D. villosum, an orthologue of TaGS5 from wheat. The SCAR marker was designated DvGS5-1443, which could successfully amplify a unique 3V-specific fragment in CD-3 and D. villosum, suggesting that this SCAR marker could facilitate targeting the chromosome 3V in the genetic background of wheat for wheat improvement

Structural organization and functional divergence of high isoelectric point α-amylase genes in bread wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.)

Abstract Background High isoelectric point α-amylase genes (Amy1) play major roles during cereal seed germination, and are associated with unacceptable high residual α-amylase activities in ripe wheat grains. However, in wheat and barley, due to extremely high homology of duplicated copies, and large and complex genome background, the knowledge on this multigene family is limited. Results In the present work, we identified a total of 41 Amy1 genes among 13 investigated grasses. By using genomic resources and experimental validation, the exact copy numbers and chromosomal locations in wheat and barley were determined. Phylogenetic and syntenic analyses revealed tandem gene duplication and chromosomal rearrangement leading to separation of Amy1 into two distinct loci, Amy1θ and Amy1λ. The divergence of Amy1λ from Amy1θ was driven by adaptive selection pressures performed on two amino acids, Arg97 and Asn233 (P > 0.95*). The predicted protein structural alteration caused by substitution of Asp233Asn in the conserved starch binding surface site, and significantly expressional differentiation during seed germination and grain development provided evidence of functional divergence between Amy1θ and Amy1λ genes. We screened out candidate copies (TaAmy1-A1/A2 and TaAmy1-D1) associated with high residual α-amylase activities in ripe grains. Furthermore, we proposed an evolutionary model for expansion dynamics of Amy1 genes. Conclusions Our study provides comprehensive analyses of the Amy1 multigene family, and defines the fixation of two spatially structural Amy1 loci in wheat and barley. Potential functional divergence between them is reflected by their sequence features and expressional patterns, and driven by gene duplication, chromosome rearrangement and natural selections during gene family evolution. Furthermore, the discrimination of differentially effective copies during seed germination and/or grain development will provide guidance to manipulation of α-amylase activity in wheat and barley breeding for better yield and processing properties

The impacts of BSMV on vegetative growth and water status in hulless barley (Hordeum vulgare var. nudum) in VIGS study

Barley stripe mosaic virus (BSMV) is an established and extensively used virus-induced gene silencing (VIGS) vector for gene function analysis in monocots. However, the phenotypes generated by targeted gene silencing may be affected or masked by symptoms of BSMV infection. To better understand the potential effects of BSMV-VIGS in hulless barley (Hordeum vulgare var. nudum), the accumulation pattern of BSMV and its impacts on vegetative growth and water status were investigated. The results indicated that the vegetative growth of infected plants was significantly and continuously impacted by BSMV from 10 to 40 days post inoculation (dpi). When the accumulation of BSMV was extremely high (7 to 11 dpi), infected plants displayed twisted leaf tips with an increased water lose rate (WLR) and decreased water content (WC). Virus accumulation declined and stabilized after 25 dpi, at this stage, the WLR and WC were unaffected in the infected plants. The efficiency of VIGS was tested by the silencing of Phytoene desaturase (PDS). RT-qPCR indicated that BSMV-VIGS can be sustained with good efficiency for up to 40 dpi under an altered condition with lower temperature (22 ±1°C) and higher relative humidity (70 ±10%). It was concluded that 25 to 40 dpi was the appropriate time zone for drought-related gene analysis by BSMV-VIGS under such condition

Dehydration induced transcriptomic responses in two Tibetan hulless barley (Hordeum vulgare var. nudum) accessions distinguished by drought tolerance

Abstract Background The harsh environment on the Qinghai-Tibetan Plateau gives Tibetan hulless barley (Hordeum vulgare var. nudum) great ability to resist adversities such as drought, salinity, and low temperature, and makes it a good subject for the analysis of drought tolerance mechanism. To elucidate the specific gene networks and pathways that contribute to its drought tolerance, and for identifying new candidate genes for breeding purposes, we performed a transcriptomic analysis using two accessions of Tibetan hulless barley, namely Z772 (drought-tolerant) and Z013 (drought-sensitive). Results There were more up-regulated genes of Z772 than Z013 under both mild (5439-VS-2604) and severe (7203-VS-3359) dehydration treatments. Under mild dehydration stress, the pathways exclusively enriched in drought-tolerance genotype Z772 included Protein processing in endoplasmic reticulum, tricarboxylic acid (TCA) cycle, Wax biosynthesis, and Spliceosome. Under severe dehydration stress, the pathways that were mainly enriched in Z772 included Carbon fixation in photosynthetic organisms, Pyruvate metabolism, Porphyrin and chlorophyll metabolism. The main differentially expressed genes (DEGs) in response to dehydration stress and genes whose expression was different between tolerant and sensitive genotypes were presented in this study, respectively. The candidate genes for drought tolerance were selected based on their expression patterns. Conclusions The RNA-Seq data obtained in this study provided an initial overview on global gene expression patterns and networks that related to dehydration shock in Tibetan hulless barley. Furthermore, these data provided pathways and a targeted set of candidate genes that might be essential for deep analyzing the molecular mechanisms of plant tolerance to drought stress

Traditional and Conditional QTL Analysis of Kernel Size- and Shape-Related Traits in Wheat (Triticum aestivum L.)

Optimal kernel size and shape were critical in improving the wheat yield potential and processing quality. A traditional and conditional QTL analysis for kernel-related traits was performed using 152 recombinant inbred lines derived from a cross between Zhongkemai 138 (ZKM138) and Kechengmai 2, whose kernel size showed significant differences. A total of 48 traditional QTLs (LOD: 3.69–14.20) were identified, with twenty-six QTLs distributed across five genomic regions. Each had at least one major stable QTL for kernel-related traits. We deduced from the co-location and conditional QTL analysis results that R3D and R4B.1 primarily controlled kernel shape, while R4D, R6A, and R6D2 primarily contributed to kernel size and the final thousand-kernel weight, potentially providing the genetic basis for the ZKM138’s high TKW and large-kernel performance. R4D may be involved with Rht2, and the possible regulatory effects of the other four QTL clusters are more likely to be influenced by unknown genes. The KASP markers validated their effect on kernel-related traits, and they were used to analyze the transmissibility and distribution of superior genotypes in ZKM138 derivatives and global wheat cultivars, respectively. These findings may serve as a reference for future genetic improvement of the ideal kernel morphology

Molecular Cytogenetic Identification of the Wheat–Dasypyrum villosum T3DL·3V#3S Translocation Line with Resistance against Stripe Rust

The annual species Dasypyrum villosum possesses several potentially valuable genes for the improvement of common wheat. Previously, we identified a new stripe rust-resistant line, the Chinese Spring (CS)–D. villosum 3V#3 (3D) substitution line (named CD-3), and mapped its potential rust resistance gene (designated as YrCD-3) on the 3V#3 chromosome originating from D. villosum. The objective of the present study was to further narrow down the YrCD-3 locus to a physical region and develop wheat-3V#3 introgression lines with strong stripe rust resistance. By treating CD-3 seeds with 60Co γ-irradiation, two CS-3V#3 translocation lines, T3V#3S.3DL and T3DS.3V#3L (termed 22-12 and 24-20, respectively), were identified from the M4 generation through a combination of non-denaturing fluorescence in situ hybridization (ND-FISH) and functional molecular markers. Stripe rust resistance tests showed that the line 22-12 exhibited strong stripe rust resistance similarly to CD-3, whereas 24-20 was susceptible to stripe rust similarly to CS, indicating that YrCD-3 is located on the short arm of 3V#3. The line 22-12 can potentially be used for further wheat improvement. Additionally, to trace 3V#3 in the wheat genetic background, we produced 30 3V#3-specific sequence tag (EST) markers, among which, 11 markers could identify 3V#3S. These markers could be valuable in fine-mapping YrCD-3

La percepción del aspecto imperfectivo del Pretérito Imperfecto : Estudio de las dificultades en alumnos suecos de español como lengua extranjera en nivel A2

Uno de los aspectos de la gramática española que presenta mayor dificultad para el aprendizaje y uso es la diferenciación entre los distintos tiempos verbales. En nuestro estudio nos hemos centrado en la percepción que alumnos suecos de E/LE en nivel A2 tienen del aspecto imperfectivo del pretérito imperfecto. Para lograr nuestro objetivo se seleccionaron 20 informantes cuya lengua materna es el sueco y cuyo único contacto con el español se da en la escuela, y se les entregaron cinco ejercicios. Los resultados a los que se ha llegado son que los alumnos tienen más dificultades en la comprensión del carácter aspectual del pretérito imperfecto que del pretérito indefinido. Por otra parte, dentro de los distintos usos del imperfecto, el que más errores produce es el uso descriptivo de la acción. Por último, si nos centramos en los tipos de verbos conjugados en el tiempo verbal relativo a nuestro estudio, los que resultan más difíciles de entender son los verbos de estado