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.

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 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 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.

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.

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 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.

Quantitative Trait Locus (QTLs) Mapping for Quality Traits of Wheat Based on High Density Genetic Map Combined With Bulked Segregant Analysis RNA-seq (BSR-Seq) Indicates That the Basic 7S Globulin Gene Is Related to Falling Number.

Numerous quantitative trait loci (QTLs) have been identified for wheat quality; however, most are confined to low-density genetic maps. In this study, based on specific-locus amplified fragment sequencing (SLAF-seq), a high-density genetic map was constructed with 193 recombinant inbred lines derived from Chuanmai 42 and Chuanmai 39. In total, 30 QTLs with phenotypic variance explained (PVE) up to 47.99% were identified for falling number (FN), grain protein content (GPC), grain hardness (GH), and starch pasting properties across three environments. Five NAM genes closely adjacent to QGPC.cib-4A probably have effects on GPC. QGH.cib-5D was the only one detected for GH with high PVE of 33.31-47.99% across the three environments and was assumed to be related to the nearest pina-D1 and pinb-D1genes. Three QTLs were identified for FN in at least two environments, of which QFN.cib-3D had relatively higher PVE of 16.58-25.74%. The positive effect of QFN.cib-3D for high FN was verified in a double-haploid population derived from Chuanmai 42 × Kechengmai 4. The combination of these QTLs has a considerable effect on increasing FN. The transcript levels of Basic 7S globulin and Basic 7S globulin 2 in QFN.cib-3D were significantly different between low FN and high FN bulks, as observed through bulk segregant RNA-seq (BSR). These QTLs and candidate genes based on the high-density genetic map would be beneficial for further understanding of the genetic mechanism of quality traits and molecular breeding of wheat.

Corrigendum: The Tryptophan decarboxylase 1 Gene From Aegilops variabilis No.1 Regulate the Resistance Against Cereal Cyst Nematode by Altering the Downstream Secondary Metabolite Contents Rather Than Auxin Synthesis.

[This corrects the article DOI: 10.3389/fpls.2018.01297.].

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.

New insights into the origin and evolution of α-amylase genes in green plants.

Gene duplication is a source of genetic materials and evolutionary changes, and has been associated with gene family expansion. Functional divergence of duplicated genes is strongly directed by natural selections such as organism diversification and novel feature acquisition. We show that, plant α-amylase gene family (AMY) is comprised of six subfamilies (AMY1-AMY6) that fell into two ancient phylogenetic lineages (AMY3 and AMY4). Both AMY1 and AMY2 are grass-specific and share a single-copy ancestor, which is derived from grass AMY3 genes that have undergone massive tandem and whole-genome duplications during evolution. Ancestral features of AMY4 and AMY5/AMY6 genes have been retained among four green algal sequences (Chrein_08.g362450, Vocart_0021s0194, Dusali_0430s00012 and Monegl_16464), suggesting a gene duplication event following Chlorophyceae diversification. The observed horizontal gene transfers between plant and bacterial AMYs, and chromosomal locations of AMY3 and AMY4 genes in the most ancestral green body (C. reinhardtii), provide evidences for the monophyletic origin of plant AMYs. Despite subfamily-specific sequence divergence driven by natural selections, the active site and SBS1 are well-conserved across different AMY isoforms. The differentiated electrostatic potentials and hydrogen bands-forming residue polymorphisms, further imply variable digestive abilities for a broad substrates in particular tissues or subcellular localizations.

The Tryptophan decarboxylase 1 Gene From Aegilops variabilis No.1 Regulate the Resistance Against Cereal Cyst Nematode by Altering the Downstream Secondary Metabolite Contents Rather Than Auxin Synthesis.

Cereal cyst nematode (CCN, Heterodera avenae) is a most important pathogen of wheat and causes tremendous yield loss annually over the world. Since the lack of resistance materials among wheat cultivars, identification and characterization of the resistance-related genes from the relatives of wheat is a necessary and efficient way. As a close relative of wheat with high resistance against CCN, Aegilops variabilis No.1 is believed to be a valuable source for wheat breeding against this devastating disease. However so far, very few resistance-associated genes have been characterized from this species. In this study, we present that the tryptophan decarboxylase genes from Ae. variabilis No.1 (AeVTDC1 and AeVTDC2) were both induced by CCN juveniles at the early stage of resistance response (30 h post-inoculation), with AeVTDC1 more sensitive to CCN infection than AeVTDC2. Silencing of AeVTDC1 led to compromised immunity to CCN with more CCN intrusion into roots; while overexpression AeVTDC1 in Nicotiana tabacum dramatically enhanced the resistance of plants by reducing the knots formed on roots. Metabolism analysis showed that the contents of secondary metabolites with activity of resistance to varied pathogens correlated with the expression level of AeVTDC1 in both Ae. variabilis No.1 and the transgenic tobacco plants. In addition, the content of IAA was not affected by either silencing or overexpressing of AeVTDC1. Hence, our research provided AeVTDC1 a valuable target that mediates resistance to CCN and root knot nematode (RKN, Meloidogyne naasi) without influencing the auxin biosynthesis.

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.

Genetic variation and phylogenetic relationship analysis of Jatropha curcas L. inferred from nrDNA ITS sequences.

Genetic variation and phylogenetic relationships among 102 Jatropha curcas accessions from Asia, Africa, and the Americas were assessed using the internal transcribed spacer region of nuclear ribosomal DNA (nrDNA ITS). The average G+C content (65.04%) was considerably higher than the A+T (34.96%) content. The estimated genetic diversity revealed moderate genetic variation. The pairwise genetic divergences (GD) between haplotypes were evaluated and ranged from 0.000 to 0.017, suggesting a higher level of genetic differentiation in Mexican accessions than those of other regions. Phylogenetic relationships and intraspecific divergence were inferred by Bayesian inference (BI), maximum parsimony (MP), and median joining (MJ) network analysis and were generally resolved. The J. curcas accessions were consistently divided into three lineages, groups A, B, and C, which demonstrated distant geographical isolation and genetic divergence between American accessions and those from other regions. The MJ network analysis confirmed that Central America was the possible center of origin. The putative migration route suggested that J. curcas was distributed from Mexico or Brazil, via Cape Verde and then split into two routes. One route was dispersed to Spain, then migrated to China, eventually spreading to southeastern Asia, while the other route was dispersed to Africa, via Madagascar and migrated to China, later spreading to southeastern Asia.

RNA-Seq Based Identification of Candidate Parasitism Genes of Cereal Cyst Nematode (Heterodera avenae) during Incompatible Infection to Aegilops variabilis.

One of the reasons for the progressive yield decline observed in cereals production is the rapid build-up of populations of the cereal cyst nematode (CCN, Heterodera avenae). These nematodes secrete so-call effectors into their host plant to suppress the plant defense responses, alter plant signaling pathways and then induce the formation of syncytium after infection. However, little is known about its molecular mechanism and parasitism during incompatible infection. To gain insight into its repertoire of parasitism genes, we investigated the transcriptome of the early parasitic second-stage (30 hours, 3 days and 9 days post infection) juveniles of the CCN as well as the CCN infected tissue of the host Aegilops variabilis by Illumina sequencing. Among all assembled unigenes, 681 putative genes of parasitic nematode were found, in which 56 putative effectors were identified, including novel pioneer genes and genes corresponding to previously reported effectors. All the 681 CCN unigenes were mapped to 229 GO terms and 200 KEGG pathways, including growth, development and several stimulus-related signaling pathways. Sixteen clusters were involved in the CCN unigene expression atlas at the early stages during infection process, and three of which were significantly gene-enriched. Besides, the protein-protein interaction network analysis revealed 35 node unigenes which may play an important role in the plant-CCN interaction. Moreover, in a comparison of differentially expressed genes between the pre-parasitic juveniles and the early parasitic juveniles, we found that hydrolase activity was up-regulated in pre J2s whereas binding activity was upregulated in infective J2s. RT-qPCR analysis on some selected genes showed detectable expression, indicating possible secretion of the proteins and putative role in infection. This study provided better insights into the incompatible interaction between H. avenae and the host plant Ae. varabilis. Moreover, RNAi targets with potential lethality were screened out and primarily validated, which provide candidates for engineering-based control of cereal cyst nematode in crops breeding.

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.

Effect of wide variation of the Waxy gene on starch properties in hull-less barley from Qinghai-Tibet plateau in China.

Granule-bound starch synthase I (GBSS I) plays an important role in the synthesis of amylose and in the determination of starch properties in barley grains. Genomic DNAs for the Waxy gene encoding GBSS I protein were sequenced from 34 barley accessions or lines from Qinghai-Tibet plateau in China, to identify Waxy gene nucleotide variations and study the roles of polymorphic sites of the Waxy gene on expression levels of Waxy transcripts and GBSS I proteins and on resulting starch properties. A total of 116 DNA polymorphic sites were identified within the barley Waxy gene, which divided the studied accessions into 11 haplotypes. Among 33 nucleotide polymorphic sites in coding regions, 5 SNPs in three exons were found to play different roles on the expression level of the Waxy transcript and the GBSS I protein and on the amylose content and starch properties. One SNP G(3935)-to-T substitution in the 10th exon in the accession Z999 (HP II-2) caused a high expression level of the Waxy transcript and the GBSS I protein and the amylose free phenotype. The other SNP alteration was a C(2453)-to-T in the fifth exon in the accession Z1191 (HP I-5), which drastically reduced the expression level of the Waxy transcript and the GBSS I protein and, finally, produced the amylose free phenotype. Three SNPs in the seventh exon in the accession Z1337 (HP I-6) did not significantly change the level of Waxy transcript, the GBSS I protein, and starch properties, except obviously reducing the breakdown value of starch viscosity and extending the peak time. A total of 84 DNA polymorphic sites were found in the noncoding regions. A 403 bp deletion at 5'UTR in the accession Z1979 (HP I-3) had low transcript level, low GBSS I protein level, and low amylose content due to the deletion of cis-acting DNA regulatory elements. A 191 bp insertion and a 15 bp insertion in the first intron and second exons, respectively, may be closely related to a higher transcript level of the Waxy gene and significant differences in some starch properties of the Waxy I DNA group as compared to the Waxy II DNA group. This study indicates the specific variations of the Waxy gene have a great effect on amylose synthesis and starch properties of hull-less barley, which could be very useful to produce new barley with variable starch properties.

Transcriptome assembly and analysis of Tibetan Hulless Barley (Hordeum vulgare L. var. nudum) developing grains, with emphasis on quality properties.

BACKGROUND: Hulless barley is attracting increasing attention due to its unique nutritional value and potential health benefits. However, the molecular biology of the barley grain development and nutrient storage are not well understood. Furthermore, the genetic potential of hulless barley has not been fully tapped for breeding. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we investigated the transcriptome features during hulless barley grain development. Using Illumina paired-end RNA-Sequencing, we generated two data sets of the developing grain transcriptomes from two hulless barley landraces. A total of 13.1 and 12.9 million paired-end reads with lengths of 90 bp were generated from the two varieties and were assembled to 48,863 and 45,788 unigenes, respectively. A combined dataset of 46,485 All-Unigenes were generated from two transcriptomes with an average length of 542 bp, and 36,278 among were annotated with gene descriptions, conserved protein domains or gene ontology terms. Furthermore, sequences and expression levels of genes related to the biosynthesis of storage reserve compounds (starch, protein, and ß-glucan) were analyzed, and their temporal and spatial patterns were deduced from the transcriptome data of cultivated barley Morex. CONCLUSIONS/SIGNIFICANCE: We established a sequences and functional annotation integrated database and examined the expression profiles of the developing grains of Tibetan hulless barley. The characterization of genes encoding storage proteins and enzymes of starch synthesis and (1-3;1-4)-ß-D-glucan synthesis provided an overview of changes in gene expression associated with grain nutrition and health properties. Furthermore, the characterization of these genes provides a gene reservoir, which helps in quality improvement of hulless barley.