Precisely mapping a major QTL for grain weight on chromosome 5B of the founder parent Chuanmai42 in the wheat-growing region of southwestern China.

KEY MESSAGE: QTgw.saas-5B was validated as a major thousand-grain weight-related QTL in a founder parent used for wheat breeding and then precisely mapped to a 0.6 cM interval. Increasing the thousand-grain weight (TGW) is considered to be one of the most important ways to improve yield, which is a core objective among wheat breeders. Chuanmai42, which is a wheat cultivar with high TGW and a high and stable yield, is a parent of more than 30 new varieties grown in southwestern China. In this study, a Chuanmai42-derived recombinant inbred line (RIL) population was used to dissect the genetic basis of TGW. A major QTL (QTgw.saas-5B) mapped to the Xgwm213-Xgwm540 interval on chromosome 5B of Chuanmai42 explained up to 20% of the phenotypic variation. Using 71 recombinants with a recombination in the QTgw.saas-5B interval identified from a secondary RIL population comprising 1818 lines constructed by crossing the QTgw.saas-5B near-isogenic line with the recurrent parent Chuannong16, QTgw.saas-5B was delimited to a 0.6 cM interval, corresponding to a 21.83 Mb physical interval in the Chinese Spring genome. These findings provide the foundation for QTgw.saas-5B cloning and its use in molecular marker-assisted breeding.

PAL-mediated SA biosynthesis pathway contributes to nematode resistance in wheat.

The pathogen cereal cyst nematode (CCN) is deleterious to Triticeae crops and is a threat to the global crop yield. Accession no. 1 of Aegilops variabilis, a relative of Triticum aestivum (bread wheat), is highly resistant to CCN. Our previous study demonstrated that the expression of the phenylalanine ammonia lyase (PAL) gene AevPAL1 in Ae. variabilis is strongly induced by CCN. PAL, the first enzyme of phenylpropanoid metabolism, is involved in abiotic and biotic stress responses. However, its role in plant-CCN interaction remains unknown. In the present study, we proved that AevPAL1 helps to confer CCN resistance through affecting the synthesis of salicylic acid (SA) and downstream secondary metabolites. The silencing of AevPAL1 increased the incidence of CCN infection in roots and decreased the accumulation of SA and phenylalanine (Phe)-derived specialized metabolites. The exogenous pre-application of SA also improved CCN resistance. Additionally, the functions of PAL in phenylpropanoid metabolism correlated with tryptophan decarboxylase (TDC) functioning in tryptophan metabolism pathways. The silencing of either AevPAL1 or AevTDC1 exhibited a concomitant reduction in the expression of both genes and the contents of metabolites downstream of PAL and TDC. These results suggested that AevPAL1, possibly in coordination with AevTDC1, positively contributes to CCN resistance by altering the downstream secondary metabolites and SA content in Ae. variabilis. Moreover, AevPAL1 overexpression significantly enhanced CCN resistance in bread wheat and did not exhibit significant negative effects on yield-related traits, suggesting that AevPAL1 is valuable for the genetic improvement of CCN resistance in bread wheat.

Genetic and transcriptomic dissection of an artificially induced paired spikelets mutant of wheat (Triticum aestivum L.).

KEY MESSAGE: Morphological, genetic and transcriptomic characterizations of an EMS-induced wheat paired spikelets (PS) mutant were performed. A novel qualitative locus WPS1 on chromosome 1D was identified. Grain yield of wheat is significantly associated with inflorescence or spike architecture. However, few genes related to wheat spike development have been identified and their underlying mechanisms are largely unknown. In this study, we characterized an ethyl methanesulfonate (EMS)-induced wheat mutant, wheat paired spikelets 1 (wps1). Unlike a single spikelet that usually develops at each node of rachis, a secondary spikelet appeared below the primary spikelet at most of the rachis nodes of wps1. The microscope observation showed that the secondary spikelet initiated later than the primary spikelet. Genetic analysis suggested that the PS of wps1 is controlled by a single dominant nuclear gene, designated WHEAT PAIRED SPIKELETS 1 (WPS1). Further RNA-seq based bulked segregant analysis and molecular marker mapping localized WPS1 in an interval of 208.18-220.92 Mb on the chromosome arm 1DL, which is different to known genes related to spike development in wheat. By using wheat omics data, TraesCS1D02G155200 encoding a HD-ZIP III transcription factor was considered as a strong candidate gene for WPS1. Transcriptomic analysis indicated that PS formation in wps1 is associated with auxin-related pathways and may be regulated by networks involving TB1, Ppd1, FT1, VRN1, etc. This study laid the solid foundation for further validation of the causal gene of WPS1 and explored its regulatory mechanism in PS formation and inflorescence development, which may benefit to kernel yield improvement of wheat based on optimization or design of spike architecture in the future.

Genetic dissection of quantitative trait loci for grain size and weight by high-resolution genetic mapping in bread wheat (Triticum aestivum L.).

KEY MESSAGE: Six major QTLs for wheat grain size and weight were identified on chromosomes 4A, 4B, 5A and 6A across multiple environments, and were validated in different genetic backgrounds. Grain size and weight are crucial components of wheat yield. Dissection of their genetic control is thus essential for the improvement of yield potential in wheat breeding. We used a doubled haploid (DH) population to detect quantitative trait loci (QTLs) for grain width (GW), grain length (GL), and thousand grain weight (TGW) in five environments. Six major QTLs, QGw.cib-4B.2, QGl.cib-4A, QGl.cib-5A.1, QGl.cib-6A, QTgw.cib-4B, and QTgw.cib-5A, were consistently identified in at least three individual environments and in best linear unbiased prediction (BLUP) datasets, and explained 5.65-34.06% of phenotypic variation. QGw.cib-4B.2, QTgw.cib-4B, QGl.cib-5A.1 and QGl.cib-6A had no effect on grain number per spike (GNS). In addition to QGl.cib-4A, the other major QTLs were further validated by using Kompetitive Allele Specific PCR (KASP) markers in different genetic backgrounds. Moreover, significant interactions between the three major GL QTLs and two major TGW QTLs were observed. Comparison analysis showed that QGl.cib-5A.1 and QGl.cib-6A are likely new loci. Notably, QGw.cib-4B.2 and QTgw.cib-4B were co-located on chromosome 4B and improved TGW by increasing only GW, unlike nearby or overlapped loci reported previously. Three genes associated with grain development within the QGw.cib-4B.2/QTgw.cib-4B interval were identified by searches on sequence similarity, spatial expression patterns, and orthologs. The major QTLs and KASP markers reported here will be useful for elucidating the genetic architecture of grain size and weight and for developing new wheat cultivars with high and stable yield.

Identification and candidate gene mining of HvSS1, a novel qualitative locus on chromosome 6H, regulating the uppermost internode elongation in barley (Hordeum vulgare L.).

KEY MESSAGE: A novel qualitative locus regulating the uppermost internode elongation of barley was identified and mapped on 6H, and the candidate gene mining was performed by employing various barley genomic resources. The stem of grass crops, such as barley and wheat, is composed of several interconnected internodes. The extent of elongation of these internodes determines stem height, and hence lodging, canopy architecture, and grain yield. The uppermost internode (UI) is the last internode to elongate. Its elongation contributes largely to stem height and facilitates spike exsertion, which is crucial for final grain yield. Despite the molecular mechanism underlying regulation of UI elongation was extensively investigated in rice, little is known in barley. In this study, we characterized a barley spontaneous mutant, Sheathed Spike 1 (SS1), showing significantly shortened UI and sheathed spike (SS). The extension of UI parenchyma cell in SS1 was significantly suppressed. Exogenous hormone treatments and RNA-seq analysis indicated that the suppression of UI elongation is possibly related to insufficient content of endogenous bioactive gibberellin. Genetic analysis showed that SS1 is possibly controlled by a qualitative dominant nuclear factor. Bulked segregant analysis and further molecular marker mapping identified a novel major locus, HvSS1, in a recombination cold spot expanding 173.44-396.33 Mb on chromosome 6H. The candidate gene mining was further conducted by analyzing sequence differences, spatiotemporal expression patterns, and variant distributions of genes in the candidate interval by employing various barley genomic resources of worldwide collections of barley accessions. This study made insight into genetic control of UI elongation in barley and laid a solid foundation for further gene cloning and functional characterization. The results obtained here also provided valuable information for similar research in wheat.

Identification and validation of two major QTLs for spike compactness and length in bread wheat (Triticum aestivum L.) showing pleiotropic effects on yield-related traits.

KEY MESSAGE: Two major and stable QTLs for spike compactness and length were detected and validated in multiple genetic backgrounds and environments, and their pleiotropic effects on yield-related traits were analyzed. Spike compactness (SC) and length (SL) are greatly associated with wheat (Triticum aestivum L.) grain yield. To detect quantitative trait loci (QTL) associated with SC and SL, two biparental populations derived from crosses of Chuanmai42/Kechengmai1 and Chuanmai42/Chuannong16 were employed to perform QTL mapping in five environments. A total of 34 QTLs were identified, in which six major QTLs were repeatedly detected in more than four environments and the best linear unbiased prediction datasets, explaining 7.13-33.6% of phenotypic variation. These major QTLs were co-located in two genomic regions on chromosome 5A and 6A, namely QSc/Sl.cib-5A and QSc/Sl.cib-6A, respectively. By developing kompetitive allele-specific PCR (KASP) markers that linked to them, the two loci were validated in different genetic backgrounds, and their interactions were also analyzed. Comparison analysis showed that QSc/Sl.cib-5A was not Vrn-A1 and Q, and QSc/Sl.cib-6A was likely a new locus for SC and SL. Both QSc/Sl.cib-5A and QSc/Sl.cib-6A had pleiotropic effects on other yield-related traits including plant height, thousand grain weight and grain length. Therefore, the two loci combined with the developed KASP markers might be potentially applicable in wheat breeding. Furthermore, based on the spatiotemporal expression patterns, gene annotation, orthologous search and sequence differences, TraesCS5A01G301400 and TraesCS6A01G090300 were considered as potential candidates for QSc/Sl.cib-5A and QSc/Sl.cib-6A, respectively. These results provided valuable information for fine mapping and cloning of the two loci in the future.

Genetic and molecular characterization of determinant of six-rowed spike of barley carrying vrs1.a4.

KEY MESSAGE: Decisive role of reduced vrs1 transcript abundance in six-rowed spike of barley carrying vrs1.a4 was genetically proved and its potential causes were preliminarily analyzed. Six-rowed spike 1 (vrs1) is the major determinant of the six-rowed spike phenotype of barley (Hordeum vulgare L.). Alleles of Vrs1 have been extensively investigated. Allele vrs1.a4 in six-rowed barley is unique in that it has the same coding sequence as Vrs1.b4 in two-rowed barley. The determinant of row-type in vrs1.a4 carriers has not been experimentally identified. Here, we identified Vrs1.b4 in two-rowed accessions and vrs1.a4 in six-rowed accessions from the Qinghai-Tibet Plateau at high frequency. Genetic analyses revealed a single nuclear gene accounting for row-type alteration in these accessions. Physical mapping identified a 0.08-cM (~ 554-kb) target interval on chromosome 2H, wherein Vrs1 was the most likely candidate gene. Further analysis of Vrs1 expression in offspring of the mapping populations or different Vrs1.b4 and vrs1.a4 lines confirmed that downregulated expression of vrs1.a4 causes six-rowed spike. Regulatory sequence analysis found a single 'TA' dinucleotide deletion in vrs1.a4 carriers within a 'TA' tandem-repeat-enriched region ~ 1 kb upstream of the coding region. DNA methylation levels did not correspond to the expression difference and therefore did not affect Vrs1 expression. More evidence is needed to verify the causal link between the 'TA' deletion and the downregulated Vrs1 expression and hence the six-rowed spike phenotype.

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

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.

Correction to: Heat stress alters genome-wide profiles of circular RNAs in Arabidopsis.

Due to an unfortunate turn of events, the first name of the fifth author appeared incorrectly in the original publication and should have read Guangbing. The correct representation of the authors' names and their affiliation is listed here and should be treated as definitive.

The draft genome of Tibetan hulless barley reveals adaptive patterns to the high stressful Tibetan Plateau.

The Tibetan hulless barley (Hordeum vulgare L. var. nudum), also called "Qingke" in Chinese and "Ne" in Tibetan, is the staple food for Tibetans and an important livestock feed in the Tibetan Plateau. The diploid nature and adaptation to diverse environments of the highland give it unique resources for genetic research and crop improvement. Here we produced a 3.89-Gb draft assembly of Tibetan hulless barley with 36,151 predicted protein-coding genes. Comparative analyses revealed the divergence times and synteny between barley and other representative Poaceae genomes. The expansion of the gene family related to stress responses was found in Tibetan hulless barley. Resequencing of 10 barley accessions uncovered high levels of genetic variation in Tibetan wild barley and genetic divergence between Tibetan and non-Tibetan barley genomes. Selective sweep analyses demonstrate adaptive correlations of genes under selection with extensive environmental variables. Our results not only construct a genomic framework for crop improvement but also provide evolutionary insights of highland adaptation of Tibetan hulless barley.

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

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.

Isolation of two new retrotransposon sequences and development of molecular and cytological markers for Dasypyrum villosum (L.).

Dasypyrum villosum is a valuable genetic resource for wheat improvement. With the aim to efficiently monitor the D. villosum chromatin introduced into common wheat, two novel retrotransposon sequences were isolated by RAPD, and were successfully converted to D. villosum-specific SCAR markers. In addition, we constructed a chromosomal karyotype of D. villosum. Our results revealed that different accessions of D. villosum showed slightly different signal patterns, indicating that distribution of repeats did not diverge significantly among D. villosum accessions. The two SCAR markers and FISH karyotype of D. villosum could be used for efficient and precise identification of D. villosum chromatin in wheat breeding.

De novo assembly and characterization of the root transcriptome of Aegilops variabilis during an interaction with the cereal cyst nematode.

BACKGROUND: Aegilops variabilis No.1 is highly resistant to cereal cyst nematode (CCN). However, a lack of genomic information has restricted studies on CCN resistance genes in Ae. variabilis and has limited genetic applications in wheat breeding. RESULTS: Using RNA-Seq technology, we generated a root transcriptome at a sequencing depth of 4.69 gigabases of Ae. variabilis No. 1 from a pooled RNA sample. The sample contained equal amounts of RNA extracted from CCN-infected and untreated control plants at three time-points. Using the Trinity method, nearly 52,081,238 high-quality trimmed reads were assembled into a non-redundant set of 118,064 unigenes with an average length of 500 bp and an N50 of 599 bp. The total assembly was 59.09 Mb of unique transcriptome sequences with average read-depth coverage of 33.25×. In BLAST searches of our database against public databases, 66.46% (78,467) of the unigenes were annotated with gene descriptions, conserved protein domains, or gene ontology terms. Functional categorization further revealed 7,408 individual unigenes and three pathways related to plant stress resistance. CONCLUSIONS: We conducted high-resolution transcriptome profiling related to root development and the response to CCN infection in Ae. variabilis No.1. This research facilitates further studies on gene discovery and on the molecular mechanisms related to CCN resistance.

Construction of a novel Wheat 55 K SNP array-derived genetic map and its utilization in QTL mapping for grain yield and quality related traits.

Wheat is one of the most important staple crops for supplying nutrition and energy to people world. A new genetic map based on the Wheat 55 K SNP array was constructed using recombinant inbred lines derived from a cross between Zhongkemai138 and Kechengmai2 to explore the genetic foundation for wheat grain features. This new map covered 2,155.72 cM across the 21 wheat chromosomes with 11,455 markers. And 2,846 specific markers for this genetic map and 148 coincident markers among different maps were documented, which was helpful for improving and updating wheat genetic and genomic information. Using this map, a total of 68 additive QTLs and 82 pairs of epistatic QTLs were detected for grain features including yield, nutrient composition, and quality-related traits by QTLNetwork 2.1 and IciMapping 4.1 software. Fourteen additive QTLs and one pair of epistatic QTLs could be detected by both software programs and thus regarded as stable QTLs here, all of which explained higher phenotypic variance and thus could be utilized for wheat grain improvement. Additionally, thirteen additive QTLs were clustered into three genomic intervals (C4D.2, C5D, and C6D2), each of which had at least two stable QTLs. Among them, C4D.2 and C5D have been attributed to the famous dwarfing gene Rht2 and the hardness locus Pina, respectively, while endowed with main effects on eight grain yield/quality related traits and epistatically interacted with each other to control moisture content, indicating that the correlation of involved traits was supported by the pleotropic of individual genes but also regulated by the gene interaction networks. Additionally, the stable additive effect of C6D2 (QMc.cib-6D2 and QTw.cib-6D2) on moisture content was also highlighted, potentially affected by a novel locus, and validated by its flanking Kompetitive Allele-Specific PCR marker, and TraesCS6D02G109500, encoding aleurone layer morphogenesis protein, was deduced to be one of the candidate genes for this locus. This result observed at the QTL level the possible contribution of grain water content to the balances among yield, nutrients, and quality properties and reported a possible new locus controlling grain moisture content as well as its linked molecular marker for further grain feature improvement.

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.

High-resolution detection of quantitative trait loci for seven important yield-related traits in wheat (Triticum aestivum L.) using a high-density SLAF-seq genetic map.

BACKGROUND: Yield-related traits including thousand grain weight (TGW), grain number per spike (GNS), grain width (GW), grain length (GL), plant height (PH), spike length (SL), and spikelet number per spike (SNS) are greatly associated with grain yield of wheat (Triticum aestivum L.). To detect quantitative trait loci (QTL) associated with them, 193 recombinant inbred lines derived from two elite winter wheat varieties Chuanmai42 and Chuanmai39 were employed to perform QTL mapping in six/eight environments. RESULTS: A total of 30 QTLs on chromosomes 1A, 1B, 1D, 2A, 2B, 2D, 3A, 4A, 5A, 5B, 6A, 6D, 7A, 7B and 7D were identified. Among them, six major QTLs QTgw.cib-6A.1, QTgw.cib-6A.2, QGw.cib-6A, QGl.cib-3A, QGl.cib-6A, and QSl.cib-2D explaining 5.96-23.75% of the phenotypic variance were detected in multi-environments and showed strong and stable effects on corresponding traits. Three QTL clusters on chromosomes 2D and 6A containing 10 QTLs were also detected, which showed significant pleiotropic effects on multiple traits. Additionally, three Kompetitive Allele Specific PCR (KASP) markers linked with five of these major QTLs were developed. Candidate genes of QTgw.cib-6A.1/QGl.cib-6A and QGl.cib-3A were analyzed based on the spatiotemporal expression patterns, gene annotation, and orthologous search. CONCLUSIONS: Six major QTLs for TGW, GL, GW and SL were detected. Three KASP markers linked with five of these major QTLs were developed. These QTLs and KASP markers will be useful for elucidating the genetic architecture of grain yield and developing new wheat varieties with high and stable yield in wheat.

Starch granule-associated proteins of hull-less barley (Hordeum vulgare L.) from the Qinghai-Tibet Plateau in China.

BACKGROUND: The starch granule-associated proteins (SGAPs) are the minor components of the starch granules and a majority of them are believed to be starch biosynthetic enzymes. The Qinghai-Tibet Plateau in China, one of the centres of origin of cultivated barley, is abundant in hull-less barley resources which exhibit high polymorphism in SGAPs. RESULTS: The SGAPs of hull-less barley from Qinghai-Tibet Plateau were analysed by one-dimensional (1-D) SDS-PAGE, 2-D PAGE and ESI-Q-TOF MS/MS. In the 1-D SDS-PAGE gel, four proteins including a 80 kDa starch synthase, actin, actin 4 and ATP synthase ß-subunit were identified as novel SGAPs. A total of six different bands were identified as starch granule-bound starch synthase I (GBSSI) and the segregation of the novel GBSSI bands in F(1) and F(2) seeds derived from yf127 × yf70 was in accordance with Mendel's law. In the 2-D PAGE gel, 92 spots were identified as 42 protein species which could be classified into 15 functional groups. Thirteen protein species were identified as SGAPs for the first time and multiple spots were identified as GBSSI. CONCLUSION: This study revealed novel SGAPs in hull-less barley from the Qinghai-Tibet Plateau in China and these will be significant in further studies of starch biosynthesis in barley.

Identification of HvLRX, a new dehydration and light responsive gene in Tibetan hulless barley (Hordeum vulgare var. nudum).

BACKGROUND: Tibetan hulless barley (Hordeum vulgare var. nudum), adjusting to the harsh environment on Qinghai-Tibet Plateau, is a good subject for analyzing drought tolerance mechanism. Several unannotated differentially expressed genes (DEGs) were identified through our previous RNA-Seq study using two hulless barley accessions with contrasting drought tolerance. One of these DEGs, HVU010048.2, showed up-regulated pattern under dehydration stress in both drought tolerant (DT) and drought susceptible (DS) accessions, while its function in drought resistance remains unknown. This new gene was named as HvLRX (light responsive X), because its expression was induced under high light intensity while suppressed under dark. OBJECTIVE: To provide preliminary bioinformatics prediction, expression pattern, and drought resistance function of this new gene. METHODS: Bioinformatics analysis of HvLRX were conducted by MEGA, PlantCARE, ProtParam, CELLO et al. The expression pattern of HvLRX under different light intensity, dehydration shock, gradual drought stress, NaCl stress, polyethylene glycol (PEG) 6000 stress and abscisic acid (ABA) treatment was investigated by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The function of HvLRX was analyzed by virus induced gene silencing (VIGS) in hulless barley and by transgenic method in tobacco. RESULTS: Full cDNAs of HvLRX were cloned and compared in three hulless barley accessions. Homologues of HvLRX protein in other plants were excavated and their phylogenetic relationship was analyzed. Several light responsive elements (ATC-motif, Box 4, G-box, Sp1, and chs-CMA1a) were identified in its promoter region. Its expression can be promoted under high light intensity, dehydration shock, gradual drought stress, PEG 6000, and NaCl stress, but was almost unchanged in ABA treatment. HvLRX-silenced plants had a higher leaf water loss rate (WLR) and a lower survival rate (SR) compared with controls under dehydration stress. The infected leaves of HvLRX-silenced plants lost their water content quickly and became withered at 10 dpi. The SR of HvLRX overexpressed transgenic tobacco plants was significantly higher than that of wild-type plants. These results indicated HvLRX play a role in drought resistance. Besides, retarded vegetative growth was detected in HvLRX-silenced hulless barley plants, which suggested that this gene is important for plant development. CONCLUSIONS: This study provided data of bioinformatics, expression pattern, and function of HvLRX. To our knowledge, this is the first report of this new dehydration and light responsive gene.

Identification and Validation of a Novel Locus Controlling Spikelet Number in Bread Wheat (Triticum aestivum L.).

Spikelet number is an important target trait for wheat yield improvement. Thus, the identification and verification of novel quantitative trait locus (QTL)/genes controlling spikelet number are essential for dissecting the underlying molecular mechanisms and hence for improving grain yield. In the present study, we constructed a high-density genetic map for the Kechengmai1/Chuanmai42 doubled haploid (DH) population using 13,068 single-nucleotide polymorphism (SNP) markers from the Wheat 55K SNP array. A comparison between the genetic and physical maps indicated high consistence of the marker orders. Based on this genetic map, a total of 27 QTLs associated with total spikelet number per spike (TSN) and fertile spikelet number per spike (FSN) were detected on chromosomes 1B, 1D, 2B, 2D, 3D, 4A, 4D, 5A, 5B, 5D, 6A, 6B, and 7D in five environments. Among them, five QTLs on chromosome 2D, 3D, 5A, and 7D were detected in multiple environments and combined QTL analysis, explaining the phenotypic variance ranging from 3.64% to 23.28%. Particularly, QTsn/Fsn.cib-3D for TSN and FSN [phenotypic variation explained (PVE) = 5.97-23.28%, limit of detection (LOD) = 3.73-18.51] is probably a novel locus and located in a 4.5-cM interval on chromosome arm 3DL flanking by the markers AX-110914105 and AX-109429351. This QTL was further validated in other two populations with different genetic backgrounds using the closely linked Kompetitive Allele-Specific PCR (KASP) marker KASP_AX-110914105. The results indicated that QTsn/Fsn.cib-3D significantly increased the TSN (5.56-7.96%) and FSN (5.13-9.35%), which were significantly correlated with grain number per spike (GNS). We also preliminary analyzed the candidate genes within this locus by sequence similarity, spatial expression patterns, and collinearity analysis. These results provide solid foundation for future fine mapping and cloning of QTsn/Fsn.cib-3D. The developed and validated KASP markers could be utilized in molecular breeding aiming to increase the grain yield in wheat.

Structural and expressional analysis of the B-hordein genes in Tibetan hull-less barley.

The B-hordein gene family was analyzed from two Tibetan hull-less barley cultivars Z09 and Z26 (Hordeum vulgare subsp. vulgare). Fourteen B-hordein genes, designated BZ09-2 to BZ09-5 (from Z09) and BZ26-1 to BZ26-10 (from Z26), were sequenced. Seven of them, similar to a previously reported BZ09-1 from Z09, were predicted to encode putative active proteins each with a signal peptide, a repetitive domain, and a C-terminal region; seven of them were predicted to be pseudogenes. The B-hordein gene family was analyzed using all known representatives of B-hordein sequences and two most similar LMW-GSs of Triticum aestivum. Alignment of these seven putative proteins with known B-hordeins and two most similar LMW-GSs of T. aestivum revealed that they shared a common motif. A large variation was observed between numbers of repeat units of predicted B-hordeins of Z26 and Z09. Phylogenetic analysis revealed that all BZ26 clones were clustered in a subgroup, and BZ09-1 formed another subgroup by itself in the putative eight active genes. In addition, six 5'-upstream regulatory sequences of the B-hordein genes were isolated from the two accessions by a single oligonucleotide nested PCR, and several different mutations were identified in the cis-acting element GLM and two distinctive sequences (Z09P-2 and Z26P-3). Phylogenetic analysis of 5'-upstream regulatory regions of the B-hordein genes showed that members from the same accession were clustered together except for two distinct members. Quantitative real time PCR analysis indicated distinct expression levels of B-hordein genes in four developing stages of developing grains in two accessions. These findings suggest B-hordein genes have intrinsic differences between accessions, and this knowledge will be useful for incorporating the B-hordeins protein in barley breeding programs.