Conventional peptides (CPs) and non-conventional peptides (NCPs) are generated from small open reading frames, but most CPs are derived from large precursors. NCPs, which are derived from sequences other than conventional open reading frames or annotated coding sequences regions, function in plant development and adaptation to stresses. Ribosome profiling, a technique for studying translational regulation, can be used to identify NCPs. Another new technique, peptidogenomics, which integrates mass spectrometry and genomics, is becoming more widely used for identifying plant NCPs. In recent years, numerous studies have investigated the roles in monocots and dicots of miRNA-derived peptides and upstream open reading frames, which have potential for improving agronomic traits. Investigating the biological functions of NCPs will advance molecular plant breeding by identifying regulators of plant growth and development. We present an overview of NCP identification methods and recent findings about NCP biological functions.

Symbiosis between soybean and rhizobia contributes to soybean yield and quality. Although secreted rhizobial type III effectors are known to regulate infection and promote nitrogen fixation, much about them remains unknown. Mutation of NopC, a type III effector from Sinorhizobium fredii HH103, reduced nodule numbers and dry weights in 310 soybean accessions, and expression of NopC in soybean hairy roots promoted symbiosis. Based on observed differences in nodule traits between Suinong 14 and Zyd 00,006 inoculated with HH103 and the NopC mutant, 11 QTL associated with rhizobia were identified in chromosome segment substitution lines (CSSLs) derived from Suinong 14 and Zyd 00006. Using chromosome fragment insertion, whole-genome sequencing of Suinong 14 and Zyd 00006, and qRT-PCR, Glyma.19G176300 (GmCRP) was identified as a candidate gene associated with NopC, and GmCRP was found to be induced by NopC to positively regulate nodulation. SNPs located in the regulatory regions of GmCRP influenced its expression response to NopC, with SNPs contributing to nodulation having been selected during domestication. Our findings reveal the function of a soybean gene encoding a rhizobial type III effector that contributes to symbiosis, and will facilitate the practical application of symbiotic nitrogen fixation in molecular breeding.

Guanine nucleotide exchange factors (GEFs) and guanine nucleotide-dissociation inhibitors (GDIs) regulate small GTPase proteins, which function as molecular switches in various signaling pathways, but their identification and functions in plants are not well understood. Using in-silico analysis and transgenic approaches, respectively, we dissected the evolutionary relationships and functions of all GEF and GDI genes in rice. Intron-exon distribution and phylogenetic analyses identified 30 GEF and 10 GDI genes in rice that shared close evolutionary relationships with other eukaryotes. Tissue-specific expression and co-expression analyses revealed that phylogenetically related genes had similar expression patterns. GEF and GDI genes were highly expressed in panicles, hulls, and stamens. Co-expression network analysis identified panicle and stamen-specific modules of co-expressed genes in both families. Mapping of these genes in known protein interactomes further identified two and one small G-protein sub-networks. A mutant library of GEF and GDI families was constructed by CRISPR knockout of each gene, and their genotypes and phenotypes were confirmed. Phenotype changes occurred with the mutation of only three genes (OsGEF5, OsGDI1, and OsGEF3). OsGEF5 and OsGDI1 single mutants exhibited significantly reduced height and longer and thinner grains, whereas OsGEF3 mutants had reduced grain length compared to the wild type. Haplotype and eGWAS analyses showed that natural variations in the three genes affected gene expression in reproductive tissues that were significantly associated with the phenotypic variation. BiFC assays demonstrated that GDI1 and GEF3 interacted with grain-size protein GS3, pointing to a role of these genes in the regulation of grain size and plant architecture connected to heterotrimeric G-proteins in rice.

Plant cell elongation depends on well-defined gene regulations, adequate nutrients, and timely cell wall modifications. Anther size is positively correlated with the number and viability of pollen grains, while little is known about molecular mechanisms underlying anther cell elongation. Here, we found that properly activated cell elongation regulators at transcriptional levels in loss-of-function ZmMs33 mutant (ms33-6038) anthers failed to promote maize anther elongation. ZmMs33 deficiency disrupted metabolic homeostasis mainly by inhibiting both photosynthesis in anther endothecium and lipid accumulation in anther tapetum. Importantly, ms33-6038 anthers displayed ectopic, premature and excessive secondary cell wall thickening in anther middle layer, which constrained cell elongation structurally and blocked nutrient flows across different anther wall layers. The metabolic disorder was only found in ms33-6038 mutant rather than several representative male-sterility lines at transcriptional and post-translational levels. Collectively, the disordered metabolisms and blocked nutrient flows defeated the activated cell elongation regulators, and finally inhibited anther elongation and growth with a unique “idling effect” in ms33-6038 mutant.

Yellow seed trait is a desirable characteristic with potential for increasing seed quality and commercial value in rapeseed, and anthocyanin and proanthocyanidins (PAs) are major seed-coat pigments. Few transcription factors involved in the regulation of anthocyanin and PAs biosynthesis have been characterized in rapeseed. In this study, we identified a transcription factor gene BnbHLH92a (BnaA06T0441000ZS) in rapeseed. Overexpressing BnbHLH92a both in Arabidopsis and in rapeseed reduced levels of anthocyanin and PAs. Correspondingly, the expression profiles of anthocyanin and PA biosynthesis genes (TT3, BAN, TT8, TT18, and TTG1) were shown by quantitative real-time PCR to be inhibited in BnbHLH92a-overexpressing Arabidopsis seeds, indicating that BnbHLH92a represses the anthocyanin and PA biosynthesis pathway in Arabidopsis. BnbHLH92a physically interacts with the BnTTG1 protein and represses the biosynthesis of anthocyanins and PAs in rapeseed. BnbHLH92a also binds directly to the BnTT18 promoter and represses its expression. These results suggest that BnbHLH92a is a novel upstream regulator of flavonoid biosynthesis in B. napus.

Rice tiller angle, as a component of plant architecture, affects rice grain yield via plant density. However, the molecular mechanism underlying rice tiller angle remains elusive. We report that the key domestication gene PROSTRATE GROWTH 1 (PROG1) controls rice tiller angle by regulating shoot gravitropism and LAZY1 (LA1)-mediated asymmetric distribution of auxin. Acting as a transcriptional repressor, PROG1 negatively regulates the expression of LA1 in light-grown rice seedlings. Overexpression of LA1 partially rescued the larger tiller angle of the PROG1 complementation transgenic plant (prog1-D). Double-mutant analysis showed that PROG1 acts upstream of LA1 to regulate shoot gravitropism and tiller angle. Mutation of Suppressors of lazy1 (SOL1), encoding DWARF3 (D3) acting in the strigolactone signal pathway, suppressed the large tiller angle of prog1-D by rescuing the transcription of LA1. The discovery of a light-sensitive PROG1-LA1 transcription regulatory module controlling rice shoot gravitropism and tiller angle sheds light on the genetic control of rice tiller angle.

Callose contributes to many biological processes of higher plants including pollen development, cell plate and vascular tissue formation, as well as regulating the transport function of plasmodesmata. The functions of callose synthase genes in maize have been little studied. We describe a maize male-sterile mutant 39 (ms39) characterized by reduced plant height. In this study, we confirmed using CRISPR/Cas9 technology that a mutation in Zm00001d043909 (ZmCals12), encoding a callose synthase, is responsible for the male sterility of the ms39 mutant. Compared with male-fertile plants, callose deposition around the dyads and tetrads in ms39 anthers was significantly reduced. Increased cell autophagy observed in ms39 anthers may have been due to the premature programmed cell death of tapetal cells, leading to collapse of the anther wall structure. Disordered glucose metabolism in ms39 may have intensified autophagy in anthers. Evaluation of the ms39 gene on maize heterosis by paired-crossed experiment with 11 maize inbred lines indicated that ms39 can be used for maize hybrid seed production.

The necrotrophic fungus Rhizoctonia cerealis is the causal agent of devastating diseases of cereal crops including wheat (Triticum aestivum). We present a high-quality genome assembly of R. cerealis Rc207, a virulent strain causing wheat sharp eyespot. The assembly (56.36 Mb) is composed of 17.87% repeat sequences and 14,433 predicted protein-encoding genes. The Rc207 genome encodes a large and diverse set of genes involved in pathogenicity, especially rich in those encoding secreted proteins, carbohydrate-active enzymes (CAZymes), peptidases, nucleases, cytochrome P450, and secondary metabolism-associated enzymes. Most secretory protein-encoding genes, including CAZymes, peroxygenases, dehydrogenases, and cytochrome P450, were up-regulated during fungal infection of wheat. We identified 831 candidate secretory effectors and validated the functions of 10 up-regulated candidate effector proteins. Of them, nine were confirmed as necrotrophic pathogen’s effectors promoting fungal infection. Abundant potential mobile or plastic genomic regions rich in repeat sequences suggest their roles in fungal adaption and virulence-associated genomic evolution. This study provides valuable resources for further comparative and functional genomics on important fungal pathogens, and provides essential tools for development of effective disease control strategies.

Cadmium (Cd) accumulation in rice grain is of health concern. Identifying genes involved in grain Cd accumulation and performing molecular breeding may reduce it. In this study, knockout of OsNRAMP2, a member of the NRAMP family, reduced grain Cd concentrations by more than 38%, and overexpressing OsNRAMP2 increased grain Cd concentrations by more than 50%. Physiological experiments showed that OsNRAMP2 facilitated Cd translocation from root to shoot by positively regulating Cd efflux from the vacuoles. At filling stage, OsNRAMP2 was highly expressed in all tissues except for husk, suggesting its role in Cd remobilization. Changes in OsNRAMP2 expression affected the concentrations of Fe, Mn, Zn, and Cu in grain and also affected rice growth. Phylogenetic analysis showed that the distribution of OsNRAMP2 haplotypes between japonica and indica was different. Among the four haplotypes of OsNRAMP2, Hap 1, with a 6-bp nucleotide insertion in exon 1, had grain Cd concentration at least 45.3% lower than any of the other three haplotypes. Almost all (99.3%) japonica accessions but rare indica accessions (4.44%) from the 3K sequenced rice genomes carry Hap 1 of OsNRAMP2. Our study sheds light on the molecular mechanism of grain Cd accumulation and provides a promising target for low-Cd rice breeding.

Heterosis and polyploidy have an overwhelming influence on plant evolution. Recently, polyploid rice hybrids have been used to breed new rice varieties because they combine the advantages of both heterosis and polyploidy. In this study, we generated six rice lines: autotetraploid rice hybrids and their autotetraploid parents, diploid donors, and hybrids of the diploid donors. To investigate the molecular mechanism controlling the effects of both hybridization and polyploidization, we performed bisulfite and RNA sequencing on young panicles at the pollen meiosis stage to compare the DNA metabolomes and transcriptomes among the six rice lines. The hybrids lines were hypermethylated compared to their corresponding parents and the autotetraploid lines showed globally increased DNA methylation of their transposable elements compared to the diploid donors. The alteration in DNA methylation level corresponded to the differential gene expressions among the rice genotypes, suggesting that methylation changes induced by polyploidization and hybridization may affect gene expression. Groups of gene candidates were identified that may be associated with heterosis and polyploidy. Our results provide DNA information that can be used to investigate epigenetic modification during heterosis and polyploidy in rice.

Grain size, characterized by a combination of grain length, width, and thickness, is one of the major determinants of yield in rice. The present study identified TATA-box binding protein-associated factor 2 (TAF2) as an essential component regulating transcription and determining grain size in rice. Map-based cloning showed that a G/T substitution in TAF2 resulted in a naturally occurring mutant called reduced grain size and plant height 1 (rgh1). The mutants, with weak edited rgh1 alleles, exhibited a small grain phenotype with reduced grain length and width, while the severe knockout mutant (rgh1-2s) was dwarf and completely sterile. Allelic test performed between rgh1 and several edited alleles confirmed that the mutation in TAF2 caused the rgh1 phenotype. GUS staining showed that TAF2 was mainly expressed in the vascular bundles of roots, stems, leaves, and grains. The cytological analysis revealed that reduced cell division in the glumes resulted in the small grain phenotype of rgh1. Further RNA-sequencing detected altered expression of genes involved in the basic biological processes in rgh1 mutant. These findings provide novel insights into the TAF2-mediated genetic mechanism regulating grain size in rice.

IQ67-domain (IQD) proteins function in plant defense and in organ development. The mechanisms by which they influence cotton fiber development are unknown. In the present study, GhIQD10 was expressed mainly in the transition period of cotton fiber development, and GhIQD10-overexpression lines showed shorter fibers. GhIQD10 interacted with GhCaM7 and the interaction was inhibited by Ca²⁺. In in vitro ovule culture, Ca²⁺ rescued the shorter-fiber phenotype of GhIQD10-overexpression lines, which were insensitive to the Ca²⁺ channel inhibitor verapamil and the Ca²⁺ pool release channel blocker 2-aminoethoxydiphenyl borate. We conclude that GhIQD10 affects cotton fiber elongation via Ca²⁺ signaling by interacting with GhCaM7. Brassinosteroid (BR) biosynthesis and signaling genes were up-regulated in GhIQD10-overexpression lines. Fiber development in these lines was not affected by epibrassinolide or the BR biosynthesis inhibitor brassinozole, indicating that the influence of GhIQD10 on fiber elongation was not associated with BR.

Although Blufensins (Bln) have important functions in the response of plants to biotic stress the precise functioning of Bln in wheat remains largely unknown. Here we isolated a Bln gene (TaBln4) from Suwon 11 infected by Puccinia striiformis f. sp. tritici (Pst). Expression of TaBln4 increased in host plants at the early stage of infection with a virulent Pst race (CYR31) but was unchanged in response to infection by an avirulent race (CYR23). Transcription levels of TaBln4 were also regulated by hormone and abiotic stresses. Expression of TaBln4 in tobacco leaves suppressed Bax-induced programmed cell death. Knockdown of TaBln4 by virus-induced gene silencing inhibited colonization of race CYR31 by increasing the accumulation of H₂O₂ and formation of hypersensitive responses (HR). Transient overexpression of TaBln4 by a transient overexpression system (BSMV-VOX) increased the susceptibility of wheat to CYR31. Results from bimolecular fluorescence complementation and pull-down assays demonstrated that TaBLN4 interacted with calmodulin. Taken together, our results suggest that TaBln4 negatively regulates resistance in wheat to Pst in a reactive oxygen species (ROS)- and HR-dependent manner.

Rapeseed (Brassica napus, AACC) was formed by hybridization between progenitor species Brassica rapa (AA) and Brassica oleracea (CC). As a result of a limited number of hybridization events between specific progenitor genotypes and strong breeding selection for oil quality traits, rapeseed has limited genetic diversity. The production of resynthesized B. napus lines via interspecific hybridization of the diploid progenitor species B. rapa and B. oleracea is one possible way to increase genetic variation in rapeseed. However, most resynthesized lines produced so far have been reported to be meiotically unstable and infertile, in contrast to established B. napus cultivars. This hinders both maintenance and use of this germplasm in breeding programs. We characterized a large set of 140 resynthesized lines produced by crosses between B. rapa and B. oleracea, as well as between B. rapa and wild C genome species (B. incana, B. hilarionis, B. montana, B. Bourgeaui, B. villosa and B. cretica) for purity (homozygosity), fertility, and genome stability. Self-pollinated seed set, seeds per ten pods as well as percentage pollen viability were used to estimate fertility. SNP genotyping was performed using the Illumina Infinium Brassica 60K array for 116 genotypes, with at least three individuals per line. Most of the material which had been advanced through multiple generations was no longer pure, with heterozygosity detected corresponding to unknown parental contributions via outcrossing. Fertility and genome stability were both genotype-dependent. Most lines had high numbers of copy number variants (CNVs), indicative of meiotic instability, and high numbers of CNVs were significantly associated with reduced fertility. Eight putatively stable resynthesized B. napus lines were observed. Further investigation of these lines may reveal the mechanisms underlying this effect. Our results suggest that selection of stable resynthesized lines for breeding purposes is possible.

Sorghum [Sorghum bicolor (L.) Moench], a multipurpose C₄ crop, is also a model species of the Poaceae family for plant research. During the process of domestication, the modification of seed dispersal mode is considered a key event, as the loss of seed shattering caused a significant increase in yield. In order to understand the seed shattering process in sorghum, we further studied eight previously identified divergent sorghum germplasm with different shattering degrees. We described their phenotypes in great detail, analyzed the histology of abscission zone, and conducted a gene co-expression analysis. We observed that the abscission layer of the most strong-shattering varieties began to differentiate before the 5-10 cm panicles development stage and was completely formed at flag leaf unfolding. The protective cells on the pedicels were also fully lignified by flowering. Through the weighted gene correlation network analysis (WGCNA), we mined for candidate genes involved in the abscission process at the heading stage. We found that these genes were mainly associated with such biological processes as hormone signal transmission (SORBI_3003G361300, SORBI_3006G216500, SORBI_3009G027800, SORBI_3007G077200), cell wall modification and degradation (SORBI_3002G205500, SORBI_3004G013800, SORBI_3010G022400, SORBI_3003G251800, SORBI_3003G254700, SORBI_3003G410800, SORBI_3009G162700, SORBI_3001G406700, SORBI_3004G042700, SORBI_3004G244600, SORBI_3001G099100), and lignin synthesis (SORBI_3004G220700, SORBI_3004G062500, SORBI_3010G214900, SORBI_3009G181800). Our study has provided candidate genes required for shedding for further study. We believe that function characterization of these genes may provide insight into our understanding of seed shattering process.

Genomic selection (GS) is a powerful tool for improving genetic gain in maize breeding. However, its routine application in large-scale breeding pipelines is limited by the high cost of genotyping platforms. Although sequencing-based and array-based genotyping platforms have been used for GS, few studies have compared prediction performance among platforms. In this study, we evaluated the predictabilities of four agronomic traits in 305 maize hybrids derived from 149 parental lines subjected to genotyping by sequencing (GBS), a 40K SNP array, and target sequence capture (TSC) using eight GS models. The GBS marker dataset yielded the highest predictabilities for all traits, followed by TSC and SNP array datasets. We investigated the effect of marker density and statistical models on predictability among genotyping platforms and found that 1K SNPs were sufficient to achieve comparable predictabilities to 10K and all SNPs, and BayesB, GBLUP, and RKHS performed well, while XGBoost performed poorly in most cases. We also selected significant SNP subsets using genome-wide association study (GWAS) analyses in three panels to predict hybrid performance. GWAS facilitated selecting effective SNP subsets for GS and thus reduced genotyping cost, but depended heavily on the GWAS panel. We conclude that there is still room for optimization of the existing SNP array, and using genotyping by target sequencing (GBTS) techniques to integrate a few functional markers identified by GWAS into the 1K SNP array holds great promise of being an effective strategy for developing desirable GS breeding arrays.

Rapeseed (Brassica napus) is an oil crop grown worldwide, making it a key plant species in molecular breeding research. However, the complexity of its polyploid genome increases sequencing costs and reduces sequencing accuracy. Target capture coupled with high-throughput sequencing is an efficient approach for detecting genetic variation at genomic regions or loci of interest. In this study, 588 resequenced accessions of rapeseed were used to develop a target capture sequencing SNP genotyping platform named BnaPan50T. The platform comprised 54,765, with 54,058 resequenced markers from the pan-genome, and 855 variant trait-associated markers for 12 agronomic traits. The capture quality of BnaPan50T was demonstrated well in 12 typical accessions. Compared with a conventional genotyping array, BnaPan50T has a high SNP density and a high proportion of SNPs in unique physical positions and in annotated functional genes, promising wide application. Target capture sequencing and whole-genome resequencing in 90 doubled-haploid lines yielded 60% specificity, 78% uniformity within tenfold coverage range, and 93% genotyping accuracy for the platform. BnaPan50T was used to construct a genetic map for quantitative trait loci (QTL) mapping, identify 21 unique QTL, and predict several candidate genes for yield-related traits in multiple environments. A set of 132 core SNP loci was selected from BnaPan50T to construct DNA fingerprints and germplasm identification resources. This study provides genomics resources to support target capture sequencing, genetic analysis and genomic breeding of rapeseed.

The development of resistant maize cultivars is the most effective and sustainable approach to combat fungal diseases. Over the last three decades, many quantitative trait loci (QTL) mapping studies reported numerous QTL for fungal disease resistance (FDR) in maize. However, different genetic backgrounds of germplasm and differing QTL analysis algorithms limit the use of identified QTL for comparative studies. The meta-QTL (MQTL) analysis is the meta-analysis of multiple QTL experiments, which entails broader allelic coverage and helps in the combined analysis of diverse QTL mapping studies revealing common genomic regions for target traits. In the present study, 128 (33.59%) out of 381 reported QTL (from 82 studies) for FDR could be projected on the maize genome through MQTL analysis. It revealed 38 MQTL for FDR (12 diseases) on all chromosomes except chromosome 10. Five MQTL namely 1_4, 2_4, 3_2, 3_4, and 5_4 were linked with multiple FDR. Total of 1910 candidate genes were identified for all the MQTL regions, with protein kinase gene families, TFs, pathogenesis-related, and disease-responsive proteins directly or indirectly associated with FDR. The comparison of physical positions of marker-traits association (MTAs) from genome-wide association studies with genes underlying MQTL interval verified the presence of QTL/candidate genes for particular diseases. The linked markers to MQTL and putative candidate genes underlying identified MQTL can be further validated in the germplasm through marker screening and expression studies. The study also attempted to unravel the underlying mechanism for FDR resistance by analyzing the constitutive gene network, which will be a useful resource to understand the molecular mechanism of defense-response of a particular disease and multiple FDR in maize.

Soybean (Glycine max L.) is a protein and oil crop grown worldwide. Its fitness may be reduced by deleterious mutations, whose identification and purging is desirable for crop breeding. In the published whole-genome re-sequenced data of 2214 soybean accessions, including 221 wild soybean, 1132 landrace cultivars and 861 improved soybean lines, we identified 115,275 deleterious single-nucleotide polymorphisms (SNPs). Numbers of deleterious alleles increased from wild soybeans to landraces and decreased from landraces to modern improved lines. Genes in selective-sweep regions showed fewer deleterious mutations than the remaining genes. Deleterious mutations explained 4.3%-48% more phenotypic variation than randomly selected SNPs for resistance to soybean cyst nematode race 2 (SCN2), soybean cyst nematode race 3 (SCN3) and soybean mosaic virus race 3 (SMV3). These findings illustrate how mutation load has shifted during soybean domestication, expansion and improvement and provide candidate sites for breeding out deleterious mutations in soybean by genome editing and/or conventional breeding focused on the selection of progeny with fewer deleterious alleles.

In maize, prolificacy, the number of ears per plant, is a trait of interest to maize breeders for breeding high grain-yielding cultivars or specialty corn, as well as being a model trait for decoding the molecular mechanism of maize evolution. Its genetic basis remains largely unknown. We identified a stable quantitative trait locus, qEN7, for ear number on chromosome 7 in both F₂ and F_2:3 populations derived from a single cross between the nonprolific inbred line Mo17 and the prolific inbred line LAN404 derived from the landrace PI217404. qEN7 explained 10.7%-11.9% of phenotypic variation, and the LAN404 allele at this locus was associated with an increase of around one ear per plant. qEN7 was confined by fine-mapping to a 0.56-Mb region containing eight annotated genes. Analysis of selection, gene expression patterns in various maize tissues, and sequence polymorphisms between the two parental lines suggested that Zm00001d020683, which encodes a putative INDETERMINATE DOMAIN (IDD) transcription factor, is the most likely candidate gene underlying qEN7. Zm00001d020683 is expressed mainly in the vegetative meristem, immature ears, and internodes and has undergone selection during maize improvement. The identification of qEN7 and the prediction of its candidate gene sheds some light on the evolution of maize ear number and provides a novel resource for breeding of multi-ear maize cultivars.

Grain size influences the yield and quality of rice (Oryza sativa L.), and grain length is one of the component traits of grain size. In this study, a near-isogenic line LB3 with long grain size was constructed using japonica rice cultivar 02428, with short grain size, as the recipient parent and indica rice cultivar ZYX, with long grain size, as the donor parent, by multi-generation backcrossing and selfing. BSA-seq was used for preliminary QTL mapping and InDel markers were developed to fine map the locus. The major QTL, tentatively named qGL10, for grain length was located in a 128.45 kb region of chromosome 10. Combined with haplotype analysis of rice varieties, expression pattern analysis of candidate genes suggested LOC_Os10g39130 (OsMADS56) as a candidate gene. Sequence alignment of OsMADS56 in 02428 and LB3 revealed that there were 15 SNPs in the promoter region and four in the coding region. Further haplotype analysis suggested that SNP9 (G/A) located in the TGTCACA motif might account for the different expression levels of OsMADS56 in 02428 and LB3. These results lay a foundation for the application of qGL10 in molecular breeding of new rice varieties.

Common wheat (Triticum aestivum L.) is the most important crop in the world and a typical allopolyploid with a large and complex genome. Pre-harvest sprouting (PHS) leads to a significant reduction in grain quality worldwide. PHS is a complex trait with related QTL located on different chromosomes. However, the study of markers and genes related to PHS resistance is limited especially for white-grained wheat. Four pairs of near isogenic lines (NILs) from a white-grained wheat cross of Chara × DM5637B*8 targeting a major QTL for PHS resistance (Qphs.ccsu-3A.1) on wheat chromosme 3AL were genotyped using the 90K SNP Illumina iSelect array. Ten SNPs were identified, with a 75%-100% consistency between genotype and phenotype in the resistant or susceptible isolines. The 10 SNPs were converted to cost-effective kompetitive allele-specific PCR (KASP) markers. Screening of 48 wheat cultivars with different phenotypes of PHS identified four KASP markers with 81.3%-85.4% conformity between genotype and phenotype. Further investigation revealed that the four SNPs (BS00022245_51, Kukri_c49927_151, BS00022884_51 and BS00110550_51) corresponding to the four validated KASP markers are residing in three independent genes (TraesCS3A03G1072800, TraesCS3A03G1072400, TraesCS3A03G1071800) close to each other with a distance of 4.28-4.48 Mb to the targeted QTL. These three annotated genes have potential functions related to PHS resistance. Our study revealed that combined use of NILs and the 90K SNP chip is a powerful approach for developing KASP markers and mining functional genes in wheat. The KASP markers for PHS resistance on chromosome 3AL are useful for high-throughput evaluation and marker-assisted selection, and the three identified genes could lead to a better understanding of the genetic pathways controlling PHS.

Grain weight and grain number are important yield component traits in wheat and identification of underlying genetic loci is helpful for improving yield. Here, we identified eight stable quantitative trait loci (QTL) for yield component traits, including five loci for thousand grain weight (TGW) and three for grain number per spike (GNS) in a recombinant inbred line population derived from cross Yangxiaomai/Zhongyou 9507 across four environments. Since grain size is a major determinant of grain weight, we also mapped QTL for grain length (GL) and grain width (GW). QTGW.caas-2D, QTGW.caas-3B, QTGW.caas-5A and QTGW.caas-7A.2 for TGW co-located with those for grain size. QTGW.caas-2D also had a consistent genetic position with QGNS.caas-2D, suggesting that the pleiotropic locus is a modulator of trade-off effect between TGW and GNS. Sequencing and linkage mapping showed that TaGL3-5A and WAPO-A1 were candidate genes of QTGW.caas-5A and QTGW.caas-7A.2, respectively. We developed Kompetitive allele specific PCR (KASP) markers linked with the stable QTL for yield component traits and validated their genetic effects in a diverse panel of wheat cultivars from the Huang-Huai River Valley region. KASP-based genotyping analysis further revealed that the superior alleles of all stable QTL for TGW but not GNS were subject to positive selection, indicating that yield improvement in the region largely depends on increased TGW. Comparative analyses with previous studies showed that most of the QTL could be detected in different genetic backgrounds, and QTGW.caas-7A.1 is likely a new QTL. These findings provide not only valuable genetic information for yield improvement but also useful tools for marker-assisted selection.

Grain size and weight are key components of wheat yield. Exploitation of major underlying quantitative trait loci (QTL) can improve yield potential in wheat breeding. A recombinant inbred line (RIL) population was constructed to detect QTL for thousand-grain weight (TGW), grain length (GL) and grain width (GW) across eight environments. Genomic regions associated with grain size and grain weight were identified on chromosomes 4A and 6A using bulked segregant exome sequencing (BSE-Seq) analysis. After constructing genetic maps, six major QTL detected in at least four individual environments and in best linear unbiased estimator (BLUE) datasets, explained 7.50%-23.45% of the phenotypic variation. Except for QGl.cib-4A, the other five QTL were co-located in two regions, namely QTgw/Gw.cib-4A and QTgw/Gw/Gl.cib-6A. Interactions of these QTL were analyzed. Unlike QTgw/Gw/Gl.cib-6A, QTgw/Gw.cib-4A and QGl.cib-4A had no effect on grain number per spike (GNS). The QTL were validated in a second cross using Kompetitive Allele Specific PCR (KASP) markers. Since QTgw/Gw.cib-4A was probably a novel locus, it and the KASP markers reported here can be used in wheat breeding. TraesCS4A03G0191200 was predicted to be potential candidate gene for QTgw/Gw.cib-4A based on the sequence differences, spatiotemporal expression patterns, gene annotation and haplotype analysis. Our findings will be useful for fine mapping and for marker-assisted selection in wheat grain yield improvement.

Genetic transformation is widely used to improve target traits and to study gene function in wheat. However, transformation efficiency depends on the physiological status of the recipient genotype and that is affected by several factors including powdery mildew (PM) infection. The widely used recipient variety Fielder is very susceptible to PM. Therefore, it would be beneficial to develop PM resistant derivatives with high regeneration ability for use in genetic transformation. In the present study PM resistant lines CB037 and Pm97033 carrying genes Pm21 and PmV, respectively, were backcrossed to Fielder with selection for PM resistance. Five lines, NT89, NT90, NT154, and WT48 with Pm21 and line FL347 with PmV were developed, identified by molecular markers and genomic in situ hybridization (GISH) or fluorescent in situ hybridization (FISH), and further subjected to detailed assessment of agronomic traits and regeneration ability following genetic transformation capacity. Lines FL347, WT48, NT89 and NT154 assessed as being equal to, or superior, to Fielder in regeneration and transformation ability are recommended as suitable materials for the replacement of Fielder for wheat gene transfer and genome editing study.

Rye (Secale cereale) is a valuable gene donor for wheat improvement, especially for its resistance to diseases. Developing rye-derived resistance sources is important for wheat breeding. In the present study, two wheat-rye derivatives, designated JS016 and JS110, were produced by crossing common wheat cultivar Yangmai 23 with Pakistani rye accession W2A. Using sequential genomic in situ hybridization (GISH) and multicolor fluorescence in situ hybridization (mc-FISH), JS016 and JS110 were identified as a T6BS.6RL translocation line and a T6BS.6BL6RL translocation line, respectively. Ten newly 6RL chromosome arm-specific markers were developed and used to confirm the 6RL translocation. The wheat 55K single-nucleotide polymorphism (SNP) array further verified the molecular cytogenetic identification results above and clarified their breakpoints at 430.9 and 523.0 Mb of chromosome 6B in JS016 and JS110, respectively. Resistance spectrum and allelism test demonstrated that JS016 and JS110 possessed novel powdery mildew resistance gene(s) that was derived from the 6RL translocation but differed from Pm20. Moreover, JS016 and JS110 had better agronomic traits than the previously reported 6RL translocation line carrying Pm20. To efficiently transfer and detect the 6RL translocation from JS016 and JS110, one 6RL-specific Kompetitive allele specific PCR (KASP) marker was developed and validated in high throughput marker-assisted selection (MAS).

Foxtail millet (Setaria italica) is an important C₄ model crop; however, due to its high-density planting and high stature, lodging at the filling stage resulted in a serious reduction in yield and quality. Therefore, it is imperative to identify and deploy the genes controlling foxtail millet plant height. In this study, we used a semi-dwarf line 263A and an elite high-stalk breeding variety, Chuang 29 to construct an F₂ population to identify dwarf genes. We performed transcriptome analysis (RNA-seq) using internode tissues sampled at three jointing stages of 263A and Chuang 29, as well as bulk segregant analysis (BSA) on their F₂ population. A total of 8918 differentially expressed genes (DEGs) were obtained from RNA-seq analysis, and GO analysis showed that DEGs were enriched in functions such as “gibberellin metabolic process” and “oxidoreductase activity”, which have previously been shown to be associated with plant height. A total 593 mutated genes were screened by BSA-seq method. One hundred and seventy-six out of the 593 mutated genes showed differential expression levels between the two parental lines, and seven genes not only showed differential expression in two or three internode tissues but also showed high genomic variation in coding regions, which indicated they play a crucial role in plant height determination. Among them, we found a gibberellin biosynthesis related GA20 oxidase gene (Seita.5G404900), which had a single-base deletion at the third exon, leading to the frameshift mutation at 263A. Cleaved amplified polymorphic sequence assay and association analysis proved the single-base deletion in Seita.5G404900 co-segregated with dwarf phenotype in two independent F₂ populations planted in entirely different environments. Taken together, the candidate genes identified in this study will help to elucidate the genetic basis of foxtail millet plant height, and the molecular marker will be useful for marker-assisted dwarf breeding.

With global warming, high-temperature (HT) stress has become a major abiotic stress for crops, in particular summer maize in China. Photosynthesis is sensitive to HT. Salicylic acid (SA) and 6-benzyladenine (6-BA) can improve the adaptation of plants to various biotic and abiotic stresses. However, their contribution to maintaining photosynthetic activity and alleviating photoinhibition in maize leaves under HT stress is still unclear. The effects of exogenous SA or 6-BA on growth, photosynthesis capacity, photosystem II (PSII) activity, subcellular ultrastructure, antioxidant system, and plant hormones in maize leaves under HT stress were investigated. Under HT conditions, application of SA or 6-BA up-regulated gibberellin and zeatin content in leaves, increasing leaf area index (LAI). It also expanded the stomata by reducing abscisic acid and jasmonic acid content in leaves, cooling them and increasing CO₂ supply to photosynthesis. A higher net photosynthetic rate, combined with increased activity of the antioxidant system, alleviated oxidative stress in maize plants sprayed with SA or 6-BA, allowing them to maintain their chloroplast ultrastructure and PSII activity, in particular electron transfer from Q_A to Q_B. The increased LAI and net photosynthetic rate per unit leaf area also resulted in the accumulation of more biomass.

Root morphology and physiology influence aboveground growth and yield formation in rice. However, root morphological and physiological differences among rice varieties with differing nitrogen (N) sensitivities and their relationship with grain yield are still unclear. In this study, rice varieties differing in N sensitivity over many years of experiments were used. A field experiment with multiple N rates (0, 90, 180, 270, and 360 kg ha⁻¹) was conducted to elucidate the effects of N application on root morphology, root physiology, and grain yield. A pot experiment with root excision and exogenous application of 6-benzyladenine (6-BA) at heading stage was used to further verify the above effects. The findings revealed that (1) under the same N application rate, N-insensitive varieties (NIV) had relatively large root biomass (root dry weight, length, and number). Grain yield was associated with root biomass in NIV. The oxidation activity and zeatin (Z) + zeatin riboside (ZR) contents in roots obviously and positively correlated with grain yield in N-sensitive varieties (NSV), and accounted for its higher grain yield than that of NIV at lower N application rates (90 and 180 kg ha⁻¹). (2) The root dry weight required for equal grain yield of NIV was greater than that of NSV. Excision of 1/10 and 1/8 of roots at heading stage had no discernible effect on the yield of Liangyoupeijiu (NIV), and it significantly reduced yield by 11.5% and 21.3% in Tianyouhuazhan (NSV), respectively, compared to the treatment without root excision. The decrease of filled kernels and grain weight after root excision was the primary cause for the yield reduction. Root excision and exogenous 6-BA application after root excision had little influence on the root activity of NIV. The oxidation activity and Z + ZR contents in roots of NSV decreased under root excision, and the increase in the proportion of excised roots aggravated these effects. The application of exogenous 6-BA increased the root activity of NSV and increased filled kernels and grain weight, thereby reducing yield loss after root excision. Thus, the root biomass of NIV was large, and there may be a phenomenon of “root growth redundancy.” Vigorous root activity was an essential feature of NSV. Selecting rice varieties with high root activity or increasing root activity by cultivation measures could lead to higher grain yield under lower N application rates.

One-time application of mixed fertilizer formed by the compounding of two controlled-release nitrogen fertilizers (CRUs) with targeted N supply during the periods from transplantation (TS) to panicle initiation (PI) and from PI to heading (HS) is expected to synchronize the double-peak N demand of rice. However, its effects on the yield and N use efficiency (NUE) of labor-intensive double-cropping rice were unknown. Two targeted CRU (CRU_A and CRU_B) were compounded in five ratios (CRU_A: CRU_B = 10:0, 7:3, 5:5, 3:7, and 0:10) to form five mixed fertilizers (BBFs): BBF1-5. A field experiment was performed to investigate the characteristics of N supply in early and late seasons under different BBFs and their effects on N uptake, yield, and ammonia volatilization (AV) loss from paddy fields of double-cropping rice. Conventional high-yield fertilization (CK, three split applications of urea) and zero-N treatments were established as controls. The N supply dropped significantly with the increased compound ratio of CRU_B during the period from TS to PI, but increased during the period from PI to HS. With the exception of the period from TS to PI in the late rice season, the N uptake of early and late rice maintained close synchronicity with the N supply of BBFs during the double-peak periods. Excessive N supply (BBF1 and BBF2) in the late rice season during the period from TS to PI increased N loss by AV. The effect of BBF on grain yield increase varied widely between seasons, irrespective of year. Among the BBFs, the BBF2 treatment of early rice not only stabilized the spikelets per panicle but also ensured a high number of effective panicles by promoting N uptake during the period from TS to PI and a high grain-filling percentage by appropriately reducing the N supply at the later PI stage, resulting in the highest rice yield. While stabilizing the effective panicle number, the BBF4 treatment of late rice increased the number of spikelets per panicle by promoting N uptake during the period from PI to HS, resulting in the highest rice yield. The two-year average yield and apparent N recovery efficiency of the BBF2 treatment during the early rice season were 9.6 t ha⁻¹ and 45.3%, while those of late rice in BBF4 were 9.6 t ha⁻¹ and 43.0%, respectively. The yield and NUE indexes of BBF2 in early rice and BBF4 in late rice showed no significant difference from those of CK. The AVs of BBF2 during the early rice season and of BBF4 during the late rice season were 50.0% and 76.8% lower, respectively, than those of CK. BBF2 and BBF4 could effectively replace conventional urea split fertilization in early and late rice seasons, ensuring rice yield and NUE and reducing AV loss in paddy fields.

Reduced photosynthesis results directly from nitrogen or water deficiency in wheat plants, and leads to a decrease in grain yield. In this study, by measuring the effects of water and N deficiencies, both individually and combined, we characterized the responses of wheat (Triticum aestivum L. Yumai 49-198) plants to these two deficiencies using physiological measurements and comparative proteomics. Significant decreases in grain yield and leaf photosynthetic performance were observed in all deficiency conditions, and 106 photosynthetic proteins that showed responses were identified. Nitrogen deficiency induced the least change in photosynthetic proteins, and similar changes in most of these proteins were also observed for the combined nitrogen and water deficiencies. Water deficiency induced the largest change in photosynthetic proteins and resulted in the lowest 1000-kernel weight. Severe decreases in photosynthesis in both the water-deficiency and combined N and water deficiency groups were reflected mainly in an imbalanced ATP/NADPH ratio associated with the light reaction, which influences carbon metabolism in the Calvin cycle. Photorespiration was respectively stimulated or inhibited by N or water deficiency, while suppression of photorespiratory flux and activation of nitrogen recycling were observed in the combined N and water deficiency treatments. Comparison of photosynthetic proteins between experimental sites suggested that precipitation affected linear electron flow in the photoreaction, and thus photosynthetic efficiency. Our results provide a baseline for future studies of the roles of these photosynthetic proteins in the response to N or water deficiency and their effect on 1000-kernel weight.

The purpose of this study was to identify the physiological mechanism underlying the effects of high temperature and waterlogging on summer maize. The stem development and yield of the maize hybrid Denghai 605 in response to high-temperature stress, waterlogging stress, and their combination applied for six days at the third-leaf, sixth-leaf, and tasseling stages were recorded. The combined stresses reduced lignin biosynthetic enzyme activity and lignin accumulation, leading to abnormal stem development. Reduction of the area and number of vascular bundles in stems led to reduced dry matter accumulation and allocation. Decreased grain dry weight at all three stages reduced grain yield relative to a control. In summary, high temperature, waterlogging, and their combined stress impaired stem development and grain yield of summer maize. The combined stresses were more damaging than either stress alone.

Only few glufosinate-tolerant genes, such as phosphinothricin acetyltransferase (PAT) and bialaphos resistance (bar) identified from Streptomyces, are currently available for developing genetically modified rice in agricultural application. Following the rapid development of genome editing technology, generation of novel glufosinate-tolerant gene resources through artificial evolution of endogenous genes is more promising and highly desirable in rice molecular breeding program. In this study, the endogenous Glutamine synthetase1 (OsGS1) was artificially evolved by base-editing-mediated gene evolution (BEMGE) in rice cells to create novel alleles conferring glufosinate tolerance in rice germplasms. Two novel glufosinate-tolerant OsGS1 alleles (OsGS1-AVPS and OsGS1-+AF) and one reported tolerant allele (OsGS1-SGTA) were successfully identified from approximately 4200 independent hygromycin-tolerant calli. Germination assays and spray tests revealed that these three OsGS1 alleles confer glufosinate tolerance in rice. Furthermore, OsGS1-AVPS and OsGS1-SGTA were quickly deployed into the elite rice cultivar Nangeng 46 through precise base editing. Overall, our results demonstrate the feasibility of developing glufosinate-tolerant rice by editing an endogenous rice gene in molecular breeding programs.

Commonly used reporters rely on a single property, such as the fluorescence of GFP and visible color of anthocyanins, therefore these reporters hardly handle the complicated condition in practice. Betaxanthins are a group of plant natural products derived from the amino acid tyrosine. Its visible yellow-orange color and green fluorescence under blue light make it a promising new reporter. Only two enzymatic reactions are required to convert tyrosine into betaxanthins. Here, we synthesized an open reading frame named Bx that contained all the betaxanthins biosynthetic genes and demonstrated its use as a powerful and efficient reporter in tobacco, carrot, and tomato.

Simultaneously improving Fusarium head blight (FHB) resistance and grain yield is challenging in wheat breeding. The correlations between spikelet compactness (SC), grain number per spike (GNS), thousand-grain weight (TGW) and FHB resistance remains unclear in common wheat. Identification of major quantitative trait loci (QTL) conferring FHB resistance and yield components, and development of breeder-friendly markers for the QTL are prerequisites for marker-assisted selection (MAS). Here, a recombinant inbred line (RIL) population derived from a cross between a resistant cultivar Yangmai 12 (YM12) and a susceptible cultivar Yanzhan 1 (YZ1) was used to map QTL for FHB resistance and yield components. A total of 22 QTL were identified; among these, six are likely to be new for corresponding traits. A QTL cluster (Qclu.yas-2D) for FHB type II resistance, SC, GNS, and TGW was detected on chromosome 2D. Breeder-friendly kompetitive allele-specific PCR (KASP) markers flanking the interval of Qclu.yas-2D were developed and validated in a diverse panel of 166 wheat cultivars and advanced lines. The YM12 alleles of Qclu.yas-2D significantly increased FHB resistance, SC, and GNS but decreased TGW in the validation population. The KASP markers developed for Qclu.yas-2D have great potential for breeding high-yielding wheat cultivars with enhanced FHB resistance.