The RING-finger ubiquitin E3 ligase TaPIR1 targets TaHRP1 for degradation to suppress chloroplast function.

Chloroplasts are key players in photosynthesis and immunity against microbial pathogens. However, the precise and timely regulatory mechanisms governing the control of photosynthesis-associated nuclear genes (PhANGs) expression in plant immunity remain largely unknown. Here we report that TaPIR1, a Pst-induced RING-finger E3 ubiquitin ligase, negatively regulates Pst resistance by specifically interacting with TaHRP1, an atypical transcription factor histidine-rich protein. TaPIR1 ubiquitinates the lysine residues K131 and K136 in TaHRP1 to regulate its stability. TaHRP1 directly binds to the TaHRP1-binding site elements within the PhANGs promoter to activate their transcription via the histidine-rich domain of TaHRP1. PhANGs expression induces the production of chloroplast-derived ROS. Although knocking out TaHRP1 reduces Pst resistance, TaHRP1 overexpression contributes to photosynthesis, and chloroplast-derived ROS production, and improves disease resistance. TaPIR1 expression inhibits the downstream activation of TaHRP1 and TaHRP1-induced ROS accumulation in chloroplasts. Overall, we show that the TaPIR1-mediated ubiquitination and degradation of TaHRP1 alters PhANGs expression to disrupt chloroplast function, thereby increasing plant susceptibility to Pst.

Multi-omic analysis reveals the effects of interspecific hybridization on the synthesis of seed reserve polymers in a Triticum turgidum ssp. durum × Aegilops sharonensis amphidiploid.

BACKGROUND: Wheat grain endosperm is mainly composed of proteins and starch. The contents and the overall composition of seed storage proteins (SSP) markedly affect the processing quality of wheat flour. Polyploidization results in duplicated chromosomes, and the genomes are often unstable and may result in a large number of gene losses and gene rearrangements. However, the instability of the genome itself, as well as the large number of duplicated genes generated during polyploidy, is an important driving force for genetic innovation. In this study, we compared the differences in starch and SSP, and analyzed the transcriptome and metabolome among Aegilops sharonensis (R7), durum wheat (Z636) and amphidiploid (Z636×R7) to reveal the effects of polyploidization on the synthesis of seed reserve polymers. RESULTS: The total starch and amylose content of Z636×R7 was significantly higher than R7 and lower than Z636. The gliadin and glutenin contents of Z636×R7 were higher than those in Z636 and R7. Through transcriptome analysis, there were 21,037, 2197, 15,090 differentially expressed genes (DEGs) in the three comparison groups of R7 vs Z636, Z636 vs Z636×R7, and Z636×R7 vs R7, respectively, which were mainly enriched in carbon metabolism and amino acid biosynthesis pathways. Transcriptome data and qRT-PCR were combined to analyze the expression levels of genes related to storage polymers. It was found that the expression levels of some starch synthase genes, namely AGP-L, AGP-S and GBSSI in Z636×R7 were higher than in R7 and among the 17 DEGs related to storage proteins, the expression levels of 14 genes in R7 were lower than those in Z636 and Z636×R7. According to the classification analysis of all differential metabolites, most belonged to carboxylic acids and derivatives, and fatty acyls were enriched in the biosynthesis of unsaturated fatty acids, niacin and nicotinamide metabolism, one-carbon pool by folate, etc. CONCLUSION: After allopolyploidization, the expression of genes related to starch synthesis was down-regulated in Z636×R7, and the process of starch synthesis was inhibited, resulting in delayed starch accumulation and prolongation of the seed development process. Therefore, at the same development time point, the starch accumulation of Z636×R7 lagged behind that of Z636. In this study, the expression of the GSe2 gene in Z636×R7 was higher than that of the two parents, which was beneficial to protein synthesis, and increased the protein content. These results eventually led to changes in the synthesis of seed reserve polymers. The current study provided a basis for a greater in-depth understanding of the mechanism of wheat allopolyploid formation and its stable preservation, and also promoted the effective exploitation of high-value alleles.

Integration of transcriptomics, metabolomics, and hormone analysis revealed the formation of lesion spots inhibited by GA and CTK was related to cell death and disease resistance in bread wheat (Triticum aestivum L.).

BACKGROUND: Wheat is one of the important grain crops in the world. The formation of lesion spots related to cell death is involved in disease resistance, whereas the regulatory pathway of lesion spot production and resistance mechanism to pathogens in wheat is largely unknown. RESULTS: In this study, a pair of NILs (NIL-Lm5W and NIL-Lm5M) was constructed from the BC1F4 population by the wheat lesion mimic mutant MC21 and its wild genotype Chuannong 16. The formation of lesion spots in NIL-Lm5M significantly increased its resistance to stripe rust, and NIL-Lm5M showed superiour agronomic traits than NIL-Lm5W under stripe rust infection.Whereafter, the NILs were subjected to transcriptomic (stage N: no spots; stage S, only a few spots; and stage M, numerous spots), metabolomic (stage N and S), and hormone analysis (stage S), with samples taken from normal plants in the field. Transcriptomic analysis showed that the differentially expressed genes were enriched in plant-pathogen interaction, and defense-related genes were significantly upregulated following the formation of lesion spots. Metabolomic analysis showed that the differentially accumulated metabolites were enriched in energy metabolism, including amino acid metabolism, carbohydrate metabolism, and lipid metabolism. Correlation network diagrams of transcriptomic and metabolomic showed that they were both enriched in energy metabolism. Additionally, the contents of gibberellin A7, cis-Zeatin, and abscisic acid were decreased in leaves upon lesion spot formation, whereas the lesion spots in NIL-Lm5M leaves were restrained by spaying GA and cytokinin (CTK, trans-zeatin) in the field. CONCLUSION: The formation of lesion spots can result in cell death and enhance strip rust resistance by protein degradation pathway and defense-related genes overexpression in wheat. Besides, the formation of lesion spots was significantly affected by GA and CTK. Altogether, these results may contribute to the understanding of lesion spot formation in wheat and laid a foundation for regulating the resistance mechanism to stripe rust.

A co-located QTL for seven spike architecture-related traits shows promising breeding use potential in common wheat (Triticum aestivum L.).

KEY MESSAGE: A co-located novel QTL for TFS, FPs, FMs, FFS, FFPs, KWS, and KWPs with potential of improving wheat yield was identified and validated. Spike-related traits, including fertile florets per spike (FFS), kernel weight per spike (KWS), total florets per spike (TFS), florets per spikelet (FPs), florets in the middle spikelet (FMs), fertile florets per spikelet (FFPs), and kernel weight per spikelet (KWPs), are key traits in improving wheat yield. In the present study, quantitative trait loci (QTL) for these traits evaluated under various environments were detected in a recombinant inbred line population (msf/Chuannong 16) mainly genotyped using the 16 K SNP array. Ultimately, we identified 60 QTL, but only QFFS.sau-MC-1A for FFS was a major and stably expressed QTL. It was located on chromosome arm 1AS, where loci for TFS, FPs, FMs, FFS, FFPs, KWS, and KWPs were also simultaneously co-mapped. The effect of QFFS.sau-MC-1A was further validated in three independent segregating populations using a Kompetitive Allele-Specific PCR marker. For the co-located QTL, QFFS.sau-MC-1A, the presence of a positive allele from msf was associate with increases for all traits: + 12.29% TFS, + 10.15% FPs, + 13.97% FMs, + 17.12% FFS, + 14.75% FFPs, + 22.17% KWS, and + 19.42% KWPs. Furthermore, pleiotropy analysis showed that the positive allele at QFFS.sau-MC-1A simultaneously increased the spike length, spikelet number per spike, and thousand-kernel weight. QFFS.sau-MC-1A represents a novel QTL for marker-assisted selection with the potential for improving wheat yield. Four genes, TraesCS1A03G0012700, TraesCS1A03G0015700, TraesCS1A03G0016000, and TraesCS1A03G0016300, which may affect spike development, were predicted in the physical interval harboring QFFS.sau-MC-1A. Our results will help in further fine mapping QFFS.sau-MC-1A and be useful for improving wheat yield.

Deacetylation of chitin oligomers by Fusarium graminearum polysaccharide deacetylase suppresses plant immunity.

Chitin is a long-chain polymer of ß-1,4-linked N-acetylglucosamine that forms rigid microfibrils to maintain the hyphal form and protect it from host attacks. Chitin oligomers are first recognized by the plant receptors in the apoplast region, priming the plant's immune system. Here, seven polysaccharide deacetylases (PDAs) were identified and their activities on chitin substrates were investigated via systematic characterization of the PDA family from Fusarium graminearum. Among these PDAs, FgPDA5 was identified as an important virulence factor and was specifically expressed during pathogenesis. ΔFgpda5 compromised the pathogen's ability to infect wheat. The polysaccharide deacetylase structure of FgPDA5 is essential for the pathogenicity of F. graminearum. FgPDA5 formed a homodimer and accumulated in the plant apoplast. In addition, FgPDA5 showed a high affinity toward chitin substrates. FgPDA5-mediated deacetylation of chitin oligomers prevented activation of plant defence responses. Overall, our results identify FgPDA5 as a polysaccharide deacetylase that can prevent chitin-triggered host immunity in plant apoplast through deacetylation of chitin oligomers.

A major QTL simultaneously increases the number of spikelets per spike and thousand-kernel weight in a wheat line.

KEY MESSAGE: A novel and stably expressed QTL QSNS.sicau-SSY-7A for spikelet number per spike in wheat without negative effects on thousand-kernel weight was identified and validated in different genetic backgrounds. Spikelet number per spike (SNS) is an important determinant of yield in wheat. In the present study, we combined bulked segregant analysis (BSA) and the wheat 660 K single-nucleotide polymorphism (SNP) array to rapidly identify genomic regions associated with SNS from a recombinant inbred line (RIL) population derived from a cross between the wheat lines S849-8 and SY95-71. A genetic map was constructed using Kompetitive Allele Specific PCR markers in the SNP-enriched region on the long arm of chromosome 7A. A major and stably expressed QTL, QSNS.sicau-SSY-7A, was detected in multiple environments. It was located in a 1.6 cM interval on chromosome arm 7AL flanked by the markers AX-109983514 and AX-109820548. This QTL explained 6.86-15.72% of the phenotypic variance, with LOD values ranging from 3.66 to 8.66. Several genes associated with plant growth and development were identified in the interval where QSNS.sicau-SSY-7A was located on the 'Chinese Spring' wheat and wild emmer reference genomes. Furthermore, the effects of QSNS.sicau-SSY-7A and WHEAT ORTHOLOG OFAPO1(WAPO1) on SNS were analyzed. Interestingly, QSNS.sicau-SSY-7A significantly increased SNS without negative effects on thousand-kernel weight, anthesis date and plant height, demonstrating its great potential for breeding aimed at improving grain yield. Taken together, these results indicate that QSNS.sicau-SSY-7A is a promising locus for yield improvement, and its linkage markers are helpful for fine mapping and molecular breeding.

Loss of ADP-glucose transporter in barley sex1 mutant caused shrunken endosperm but with elevated protein and ß-glucan content in whole meal.

Grain shape and plumpness affect barley yield. Despite numerous studies on shrunken endosperm mutants in barley, their molecular mechanism and application potential in the food industry are largely unknown. Here, map-based cloning, co-segregation analyses, and allelic variant validation revealed that the loss of HORVU6Hr1G037950 encoding an ADP-glucose transporter caused the shrunken endosperm in sex1. Haplotype analysis suggested that hap4 in the promoter sequence was positively related to the hundred-grain weight showing a breeding potential. A pair of near-isogenic lines targeting HORVU6Hr1G037950 was produced and characterized to investigate molecular mechanisms that SEX1 regulates endosperm development. Results presented that the absence of the SEX1 gene led to the decrease of starch content and A-type granules size, the increase of ß-glucan, protein, gelatinization temperature, soluble sugar content, amylopectin A chains, and B1 chains. Enzymatic activity, transcriptome and metabolome analyses revealed the loss of SEX1 results in an impaired ADP-glucose-to-starch conversion process, consequently leading to higher soluble sugar contents and lower starch accumulation, thereby inducing a shrunken-endosperm phenotype in sex1. Taken together, this study provides new insights into barley grain development, and the elevated protein and ß-glucan contents of the whole meal in sex1 imply its promising application in the food industry.

A promising QTL QSns.sau-MC-3D.1 likely superior to WAPO1 for the number of spikelets per spike of wheat shows no adverse effects on yield-related traits.

KEY MESSAGE: A likely new locus QSns.sau-MC-3D.1 associated with SNS showing no negative effect on yield-related traits compared to WAPO1 was identified and validated in various genetic populations under multiple environments. The number of spikelets per spike (SNS) is one of the crucial factors determining wheat yield. Thus, improving our understanding of the genes that regulate SNS could help develop wheat varieties with higher yield. In this study, a recombinant inbred line (RIL) population (MC) containing 198 lines derived from a cross between msf and Chuannong 16 (CN16) was used to construct a genetic linkage map using the GenoBaits Wheat 16 K Panel. The genetic map contained 5,991 polymorphic SNP markers spanning 2,813.25 cM. A total of twelve QTL for SNS were detected, and two of them, i.e., QSns.sau-MC-3D.1 and QSns.sau-MC-7A, were stably expressed. QSns.sau-MC-3D.1 had high LOD values ranging from 4.99 to 11.06 and explained 9.71-16.75% of the phenotypic variation. Comparison of QSns.sau-MC-3D.1 with previously reported SNS QTL suggested that it is likely a novel one, and two kompetitive allele-specific PCR (KASP) markers were further developed. The positive effect of QSns.sau-MC-3D.1 was also validated in three biparental populations and a diverse panel containing 388 Chinese wheat accessions. Genetic analysis indicated that WHEAT ORTHOLOG OFAPO1 (WAPO1) was a candidate gene for QSns.sau-MC-7A. Pyramiding of QSns.sau-MC-3D.1 and WAP01 had a great additive effect increasing SNS by 7.10%. Correlation analysis suggested that QSns.sau-MC-3D.1 was likely independent of effective tiller number, plant height, spike length, anthesis date, and thousand kernel weight. However, the H2 haplotype of WAPO1 may affect effective tiller number and plant height. These results indicated that utilization of QSns.sau-MC-3D.1 should be given priority for wheat breeding. Geographical distribution analysis showed that the positive allele of QSns.nsau-MC-3D.1 was dominant in most wheat-producing regions of China, and it has been positively selected among modern cultivars released in China since the 1940s. Gene prediction, qRT-PCR analysis, and sequence alignment suggested that TraesCS3D03G0216800 may be the candidate gene of QSns.nsau-MC-3D.1. Taken together, these results enrich our understanding of the genetic basis of wheat SNS and will be useful for fine mapping and cloning of the gene underlying QSns.sau-MC-3D.1.

TaRBP1 stabilizes TaGLTP and negatively regulates stripe rust resistance in wheat.

The dynamic balance and distribution of sphingolipid metabolites modulate the level of programmed cell death and plant defence. However, current knowledge is still limited regarding the molecular mechanism underlying the relationship between sphingolipid metabolism and plant defence. In this study, we identified a wheat RNA-binding protein 1 (TaRBP1) and TaRBP1 mRNA accumulation significantly decreased in wheat after infection by Puccinia striiformis f. sp. tritici (Pst). Knockdown of TaRBP1 via virus-induced gene silencing conferred strong resistance to Pst by enhancing host plant reactive oxygen species (ROS) accumulation and cell death, indicating that TaRBP1 may act as a negative regulator in response to Pst. TaRBP1 formed a homopolymer and interacted with TaRBP1 C-terminus in plants. Additionally, TaRBP1 physically interacted with TaGLTP, a sphingosine transfer protein. Knockdown of TaGLTP enhanced wheat resistance to the virulent Pst CYR31. Sphingolipid metabolites showed a significant accumulation in TaGLTP-silenced wheat and TaRBP1-silenced wheat, respectively. In the presence of the TaRBP1 protein, TaGLTP failed to be degraded in a 26S proteasome-dependent manner in plants. Our results reveal a novel susceptible mechanism by which a plant fine-tunes its defence responses by stabilizing TaGLTP accumulation to suppress ROS and sphingolipid accumulation during Pst infection.

Identification and validation of two major QTLs for spikelet number per spike in wheat (Triticum aestivum L.).

The total number of spikelets (TSPN) and the number of fertile spikelets (FSPN) affect the final number of grains per spikelet in wheat. This study constructed a high-density genetic map using 55K single nucleotide polymorphism (SNP) arrays from a population of 152 recombinant inbred lines (RIL) from crossing the wheat accessions 10-A and B39. Twenty-four quantitative trait loci (QTLs) for TSPN and 18 QTLs for FSPN were localized based on the phenotype in 10 environments in 2019-2021. Two major QTLs, QTSPN/QFSPN.sicau-2D.4 (34.43-47.43 Mb) and QTSPN/QFSPN.sicau-2D.5(32.97-34.43 Mb), explained 13.97%-45.90% of phenotypic variation. Linked kompetitive allele-specific PCR (KASP) markers further validated these two QTLs and revealed that QTSPN.sicau-2D.4 had less effect on TSPN than QTSPN.sicau-2D.5 in 10-A×BE89 (134 RILs) and 10-A×Chuannong 16 (192 RILs) populations, and one population of Sichuan wheat (233 accessions). The alleles combination haplotype 3 with the allele from 10-A of QTSPN/QFSPN.sicau-2D.5 and the allele from B39 of QTSPN.sicau-2D.4 resulted in the highest number of spikelets. In contrast, the allele from B39 for both loci resulted in the lowest number of spikelets. Using bulk-segregant analysis-exon capture sequencing, six SNP hot spots that included 31 candidate genes were identified in the two QTLs. We identified Ppd-D1a from B39 and Ppd-D1d from 10-A and further analyzed Ppd-D1 variation in wheat. These results identified loci and molecular markers with potential utility for wheat breeding and laid a foundation for further fine mapping and cloning of the two loci.

Comparative analysis of Fusarium crown rot resistance in synthetic hexaploid wheats and their parental genotypes.

BACKGROUND: Fusarium crown rot (FCR) is a chronic disease of cereals worldwide. Compared with tetraploid wheat, hexaploid wheat is more resistant to FCR infection. The underlying reasons for the differences are still not clear. In this study, we compared FCR responses of 10 synthetic hexaploid wheats (SHWs) and their tetraploid and diploid parents. We then performed transcriptome analysis to uncover the molecular mechanism of FCR on these SHWs and their parents. RESULTS: We observed higher levels of FCR resistance in the SHWs compared with their tetraploid parents. The transcriptome analysis suggested that multiple defense pathways responsive to FCR infection were upregulated in the SHWs. Notably, phenylalanine ammonia lyase (PAL) genes, involved in lignin and salicylic acid (SA) biosynthesis, exhibited a higher level of expression to FCR infection in the SHWs. Physiological and biochemical analysis validated that PAL activity and SA and lignin contents of the stem bases were higher in SHWs than in their tetraploid parents. CONCLUSION: Overall, these findings imply that improved FCR resistance in SHWs compared with their tetraploid parents is probably related to higher levels of response on PAL-mediated lignin and SA biosynthesis pathways.

Identification of a suppressor for the wheat stripe rust resistance gene Yr81 in Chinese wheat landrace Dahongpao.

KEY MESSAGE: Combined with BSE-Seq analysis and multiple genetic populations, three genes involved in stripe rust resistance were identified in Chinese wheat landrace Dahongpao, including a novel suppressor on 2BS. Dahongpao (DHP), a landrace of hexaploid wheat in China, exhibits a high degree of stripe rust resistance in the field for many years. In this study, bulked segregant analysis coupled with exome capture sequencing (BSE-Seq) was used to identify genes encoding stripe rust resistance in multiple genetic populations from the cross between DHP and a susceptible hexaploid Australian cultivar, Avocet S (AvS). The most effective QTL in DHP was Yr18, explaining up to 53.08% of phenotypic variance in the F2:3 families. To identify additional genes, secondary mapping populations SP1 and SP2 were produced by crossing AvS with two resistant lines derived from F2:3 families lacking Yr18. An all-stage resistance gene, Yr.DHP-6AS, was identified via BSE-Seq analysis of SP1. Combined the recombinant plants from both SP1 and SP2, Yr.DHP-6AS was located between KP6A_1.66 and KP6A_8.18, corresponding to the same region as Yr81. In addition, secondary mapping populations SP3 and SP4 were developed by selfing a segregating line from F2:3 families lacking Yr18. A novel suppressor gene on chromosome 2BS was identified from DHP for effectively suppressing the resistance of Yr.DHP-6AS in the SP3 and SP4. As a result, the wheat lines carrying both Yr18 and Yr.DHP-6AS show higher level of stripe rust resistance than DHP, providing an effective and simple combination for developing new wheat cultivars with ASR and APR genes. Further, the newly developed KASP markers, KP6A_1.99 and KP6A_5.22, will facilitate the application of Yr.DHP-6AS in wheat breeding via marker-assisted selection.

Major and stably expressed QTL for traits related to the mature wheat embryo independent of kernel size.

KEY MESSAGE: Two major and stably expressed QTL for traits related to mature wheat embryo independent of kernel size were identified and validated in a natural population that contained 171 Sichuan wheat accessions and 49 Sichuan wheat landraces. As the juvenile of a highly differentiated plant, mature wheat (Triticum aestivum L.) embryos are highly significant to agricultural production. To understand the genetic basis of traits related to wheat embryo size, the embryo of mature kernels in a recombination inbred line that contained 126 lines from four environments was measured. The genetic loci of embryo size, including embryo length (EL), embryo width (EW), embryo area (EA), embryo length/kernel length (EL/KL), embryo width/kernel width (EW/KW), and EL/EW, were identified based on a genetic linkage map constructed based on PCR markers and the Wheat 55 K single nucleotide polymorphism (SNP) array. A total of 50 quantitative trait loci (QTL) for traits related to wheat embryo size were detected. Among them, QEL.sicau-2SY-4A for EL and QEW.sicau-2SY-7B for EW were major and stably expressed and were genetically independent of KL and KW, respectively. Their effects were further verified in a natural population that contained 171 Sichuan wheat accessions and 49 Sichuan wheat landraces. Further analysis showed that TraesCS4A02G343300 and TraesCS7B02G006800 could be candidate genes for QEL.sicau-2SY-4A and QEW.sicau-2SY-7B, respectively. In addition, significant positive correlations between EL and kernel-related traits and the 1,000-grain weight were detected. Collectively, this study broadens our understanding of the genetic basis of wheat embryo size and will be helpful for the further fine-mapping of interesting loci in the future.

A CRISPR/Cas9 Protocol for Target Gene Editing in Barley.

Previous studies of gene function rely on the existing natural genetic variation or on induction of mutations by physical or chemical mutagenesis. The availability of alleles in nature, and random mutagenesis induced by physical or chemical means, limits the depth of research. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system provides the means to rapidly modify genomes in a precise and predictable way, making it possible to modulate gene expression and modify the epigenome. Barley is the most appropriate model species for functional genomic analysis of common wheat. Therefore, the genome editing system of barley is very important for the study of wheat gene function. Here we detail a protocol for barley gene editing. The effectiveness of this method has been confirmed in our previous published studies.

Aegilops sharonensis HMW-GSs with unusually large molecular weight improves bread-making quality in wheat-Ae. sharonensis introgression lines.

BACKGROUND: Eighteen wheat (Triticum aestivum-Aegilops sharonensis) introgression lines were generated in the previous study. These lines possessed four types of high molecular weight glutenin subunit (HMW-GS) combinations consisting of one glutenin from Ae. sharonensis (Glu-1Ssh ) plus one or more HMW-GSs from common wheat (Glu-A1, Glu-B1, or Glu-D1). RESULTS: In this study, we conducted quality tests to explore the effects of 1Ssh x2.3 and 1Ssh y2.9 on the processing quality of 18 wheat-Aegilops sharonensis introgression lines. Our data showed that the 1Ssh x2.3 and 1Ssh y2.9 subunits had a positive effect on gluten and baking quality. The bread volume of all these lines was higher than that of the parental wheat line LM3. In these lines, the HMW-GS content and the HMW/LMW ratio of 66-36-11 were higher than those of LM3, and the 66-36-11 line exhibited greatly improved quality parameters in comparison with the parent LM3. CONCLUSION: These results demonstrated that the 1Ssh x2.3 and 1Ssh y2.9 subunits from Ae. sharonensis contributed immensely to gluten strength and bread-baking quality, and proved a positive relationship between the HMW-GS sizes and their effects on dough strength in vivo. The materials developed could be used by breeding programs aiming to increase bread-making quality. © 2022 Society of Chemical Industry.

HvGBSSI mutation at the splicing receptor site affected RNA splicing and decreased amylose content in barley.

Granule-bound starch synthase I (HvGBSSI) is encoded by the barley waxy (Wx-1) gene and is the sole enzyme in the synthesis of amylose. Here, a Wx-1 mutant was identified from an ethyl methane sulfonate (EMS)-mutagenized barley population. There were two single-base mutations G1086A and A2424G in Wx-1 in the mutant (M2-1105). The G1086A mutation is located at the 3' splicing receptor (AG) site of the fourth intron, resulting in an abnormal RNA splicing. The A2424G mutation was a synonymous mutation in the ninth intron. The pre-mRNA of Wx-1 was incorrectly spliced and transcribed into two abnormal transcripts. The type I transcript had a 6 bp deletion in the 5' of fifth exon, leading to a translated HvGBSSI protein lacking two amino acids with a decreased starch-binding capacity. In the type II transcript, the fourth intron was incorrectly cleaved and retained, resulting in the premature termination of the barley Wx-1 gene. The mutations in the Wx-1 decreased the enzymatic activity of the HvGBSSI enzyme and resulted in a decreased level in amylose content. This work sheds light on a new Wx-1 gene inaction mechanism.

Fine mapping of the Hairy glume (Hg) gene in a chromosome variation region at the distal terminus of 1AS.

Trichomes are differentiated epidermal cells and exist on above-ground organs of nearly all land plants with important roles in resistance to a wide range of biotic and abiotic stresses. We attempted to obtain candidate gene (s) for Hairy glume (Hg), responsible for the trichome on wheat glume, by using bulked segregant exome capture sequencing (BSE-Seq), while Hg was only mapped in 0.52-3.26 Mb of 1AS. To further fine map this gene and identify candidate genes in this region, a near isogenic line-derived population consisting of 2,050 F2 lines was generated in the present study. By analyzing this population, Hg was fine mapped into a 0.90 cM region covering a physical distance of ~825.03 Kb encompassing 6 high- and 23 low-confidence genes in the reference genome of Chinese Spring. A presence-absence variation was identified in the fine mapping region through analyses of sequence-tagged sites markers and genome sequences of the hairy glume parent of the near isogenic lines. The results presented here will be useful for further cloning Hg in wheat.

A major quantitative trait locus for wheat total root length associated with precipitation distribution.

Optimizing root system architecture (RSA) allows crops to better capture water and nutrients and adapt to harsh environment. Parental reproductive environment (PRE) has been reported to significantly affect growth and development throughout the life cycle of the next generation. In this study, 10 RSA-related traits were evaluated in seedling stage from five independent hydroponic tests using seeds harvested from five different PREs. Based on the Wheat55K SNP array-based genetic map, quantitative trait loci (QTL) for these traits were detected in a recombinant inbred line population. Twenty-eight putative QTL for RSA-related traits were detected, covering thirteen chromosomal regions. A major QTL, QTrl.sicau-2SY-4D for total root length (TRL), which was likely independent of PREs, explained 15.81-38.48% of phenotypic variations and was located at 14.96-19.59 Mb on chromosome arm 4DS. Interestingly, it showed pleiotropic effects on TRL, root area, root volume, root forks, root dry weight, and shoot dry weight. The functional marker KASP-Rht-D1 for Rht-D1 was used to genotype 2SY population and remapping QTL for TRL showed that QTrl.sicau-2SY-4D was not linked to Rht-D1. The kompetitive allele-specific PCR (KASP) marker, KASP-AX-110527441 linked to this major QTL, was developed and used to successfully validate its effect in three different genetic populations. Further analysis suggested that the positive allele at QTrl.sicau-2SY-4D was mainly utilized in wheat breeding of northwest China where precipitation was significantly lower, indicating that wheat requires longer TRL to capture water and nutrients in arid or semi-arid regions due to deficient precipitation. Additionally, four genes (TraesCS4D03G0059800, TraesCS4D03G0057800, TraesCS4D03G0064000, and TraesCS4D03G0064400) possibly related to root development were predicted in physical interval of QTrl.sicau-2SY-4D. Taken together, these results enrich our understanding on the genetic basis of RSA and provide a potentially valuable TRL QTL for wheat breeding.

Increasing the Grain Yield and Grain Protein Content of Common Wheat (Triticum aestivum) by Introducing Missense Mutations in the Q Gene.

Grain yield (GY) and grain protein content (GPC) are important traits for wheat breeding and production; however, they are usually negatively correlated. The Q gene is the most important domestication gene in cultivated wheat because it influences many traits, including GY and GPC. Allelic variations in the Q gene may positively affect both GY and GPC. Accordingly, we characterized two new Q alleles (Qs1 and Qc1-N8) obtained through ethyl methanesulfonate-induced mutagenesis. Compared with the wild-type Q allele, Qs1 contains a missense mutation in the sequence encoding the first AP2 domain, whereas Qc1-N8 has two missense mutations: one in the sequence encoding the second AP2 domain and the other in the microRNA172-binding site. The Qs1 allele did not significantly affect GPC or other processing quality parameters, but it adversely affected GY by decreasing the thousand kernel weight and grain number per spike. In contrast, Qc1-N8 positively affected GPC and GY by increasing the thousand kernel weight and grain number per spike. Thus, we generated novel germplasm relevant for wheat breeding. A specific molecular marker was developed to facilitate the use of the Qc1-N8 allele in breeding. Furthermore, our findings provide useful new information for enhancing cereal crops via non-transgenic approaches.