Identification and characterization of QSFS.sau-MC-5A for sterile florets genetically independent of fertile ones per spike in wheat.

KEY MESSAGE: A major and stable QTL for sterile florets per spike and sterile florets per spikelet was identified, it was mapped within a 2.22-Mb interval on chromosome 5AL, and the locus was validated using two segregating populations with different genetic backgrounds. Both the number of fertile florets per spike (FFS) and the number of sterile florets per spike (SFS) significantly influence the final yield of wheat (Triticum aestivum L.), and a trade-off theoretically exists between them. To enhance crop yield, wheat breeders have historically concentrated on easily measurable traits such as FFS, spikelets per spike, and spike length. Other traits of agronomic importance, including SFS and sterile florets per spikelet (SFPs), have been largely overlooked. In the study, reported here, genetic bases of SFS and SFPs were investigated based on the assessment of a population of recombinant inbred lines (RILs) population. The RIL population was developed by crossing a spontaneous mutant with higher SFS (msf) with the cultivar Chuannong 16. A total of 10 quantitative trait loci (QTL) were identified, with QSFS.sau-MC-5A for SFS and QSFPs.sau-MC-5A for SFPs being the major and stable ones, and they were co-located on the long arm of chromosome 5A. The locus was located within a 2.22-Mb interval, and it was further validated in two additional populations based on a tightly linked Kompetitive Allele-Specific PCR (KASP) marker, K_sau_5A_691403852. Expression differences and promoter sequence variations were observed between the parents for both TraesCS5A03G1247300 and TraesCS5A03G1250300. The locus of QSFS.sau-MC-5A/QSFPs.sau-MC-5A showed a significantly positive correlation with spike length, florets in the middle spikelet, and total florets per spike, but it showed no correlation with either kernel number per spike (KNS) or kernel weight per spike. Appropriate nitrogen fertilizer application led to reduced SFS and increased KNS, supporting results from previous reports on the positive effect of nitrogen fertilizer on wheat spike and floret development. Based on these results, we propose a promising approach for breeding wheat cultivars with multiple fertile florets per spike, which could increase the number of kernels per spike and potentially improve yield. Collectively, these findings will facilitate further fine mapping of QSFS.sau-MC-5A/QSFPs.sau-MC-5A and be instrumental in strategies to increase KNS, thereby enhancing wheat yield.

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.

Identification of candidate genes for adult plant stripe rust resistance transferred from Aegilops ventricosa 2NvS into wheat via fine mapping and transcriptome analysis.

KEY MESSAGE: An adult plant gene for resistance to stripe rust was narrowed down to the proximal one-third of the 2NvS segment translocated from Aegilops ventricosa to wheat chromosome arm 2AS, and based on the gene expression analysis, two candidate genes were identified showing a stronger response at the adult plant stage compared to the seedling stage. The 2NvS translocation from Aegilops ventricosa, known for its resistance to various diseases, has been pivotal in global wheat breeding for more than three decades. Here, we identified an adult plant resistance (APR) gene in the 2NvS segment in wheat line K13-868. Through fine mapping in a segregating near-isogenic line (NIL) derived population of 6389 plants, the candidate region for the APR gene was narrowed down to between 19.36 Mb and 33 Mb in the Jagger reference genome. Transcriptome analysis in NILs strongly suggested that this APR gene conferred resistance to stripe rust by triggering plant innate immune responses. Based on the gene expression analysis, two disease resistance-associated genes within the candidate region, TraesJAG2A03G00588940 and TraesJAG2A03G00590140, exhibited a stronger response to Puccinia striiformis f. sp. tritici (Pst) infection at the adult plant stage than at the seedling stage, indicating that they could be potential candidates for the resistance gene. Additionally, we developed a co-dominant InDel marker, InDel_31.05, for detecting this APR gene. Applying this marker showed that over one-half of the wheat varieties approved in 2021 and 2022 in Sichuan province, China, carry this gene. Agronomic trait evaluation of NILs indicated that the 2NvS segment effectively mitigated the negative effects of stripe rust on yield without affecting other important agronomic traits. This study provided valuable insights for cloning and breeding through the utilization of the APR gene present in the 2NvS segment.

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.

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.

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.

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.

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.

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.

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.

KRS-Net: A Classification Approach Based on Deep Learning for Koi with High Similarity.

As the traditional manual classification method has some shortcomings, including high subjectivity, low efficiency, and high misclassification rate, we studied an approach for classifying koi varieties. The main contributions of this study are twofold: (1) a dataset was established for thirteen kinds of koi; (2) a classification problem with high similarity was designed for underwater animals, and a KRS-Net classification network was constructed based on deep learning, which could solve the problem of low accuracy for some varieties that are highly similar. The test experiment of KRS-Net was carried out on the established dataset, and the results were compared with those of five mainstream classification networks (AlexNet, VGG16, GoogLeNet, ResNet101, and DenseNet201). The experimental results showed that the classification test accuracy of KRS-Net reached 97.90% for koi, which is better than those of the comparison networks. The main advantages of the proposed approach include reduced number of parameters and improved accuracy. This study provides an effective approach for the intelligent classification of koi, and it has guiding significance for the classification of other organisms with high similarity among classes. The proposed approach can be applied to some other tasks, such as screening, breeding, and grade sorting.

Genome-Wide Survey and Functional Verification of the NAC Transcription Factor Family in Wild Emmer Wheat.

The NAC transcription factor (TF) family is one of the largest TF families in plants, which has been widely reported in rice, maize and common wheat. However, the significance of the NAC TF family in wild emmer wheat (Triticum turgidum ssp. dicoccoides) is not yet well understood. In this study, a genome-wide investigation of NAC genes was conducted in the wild emmer genome and 249 NAC family members (TdNACs) were identified. The results showed that all of these genes contained NAM/NAC-conserved domains and most of them were predicted to be located on the nucleus. Phylogenetic analysis showed that these 249 TdNACs can be classified into seven clades, which are likely to be involved in the regulation of grain protein content, starch synthesis and response to biotic and abiotic stresses. Expression pattern analysis revealed that TdNACs were highly expressed in different wheat tissues such as grain, root, leaves and shoots. We found that TdNAC8470 was phylogenetically close to NAC genes that regulate either grain protein or starch accumulation. Overexpression of TdNAC8470 in rice showed increased grain starch concentration but decreased grain Fe, Zn and Mn contents compared with wild-type plants. Protein interaction analysis indicated that TdNAC8470 might interact with granule-bound starch synthase 1 (TdGBSS1) to regulate grain starch accumulation. Our work provides a comprehensive understanding of the NAC TFs family in wild emmer wheat and establishes the way for future functional analysis and genetic improvement of increasing grain starch content in wheat.