Wheat is one of the most important food crops in the world, and grain number per spike (GNS) is one of the most important factors affecting wheat grain yield. Therefore, identifying genes controlling GNS is important for wheat production. However, wheat is an allohexaploid species and has a highly complex genome, and the isolation of wheat genes is challenging. In this study, we identified a candidate gene, TraesCS4B02G047100 (TaRLK-4B), associated with GNS using both quantitative trait locus (QTL) mapping and genome-wide association study (GWAS) based on RNA-Seq. We obtained three homozygous mutant lines, bb-1, bb-2 and bb-3, using the CRISPR/Cas9 gene editing system. Compared with Fielder (wild type, WT), the three mutant lines exhibited significant reductions in GNS, total spikelets per spike (TSS) and spike length (SL). These results indicated that TaRLK-4B positively regulates GNS and its related traits TSS and SL. The TaRLK-4B gene contains two exons and one intron and belongs to the largest subfamily of LRR-RLKs. We performed RNA-Seq analysis using spikes from the WT and bb-2 mutant line at the tillering stage. A total of 1,193 differentially expressed genes (DEGs) were identified, including previously cloned TaSPL17 homologous genes in the three subgenomes and their orthologous gene OsIPA1 (OsSPL14) in rice. Combining RNA-Seq data of TaRLK-4B and DAP-Seq data of the TaSPL17-7D, we identified 136 overlapping genes which are likely downstream targets of TaSPL17-7D. Therefore, we hypothesized that TaRLK-4B represents a novel putative component of the TaSPL17-centered regulatory network. In addition, haplotype analysis revealed that Haplotype 2 (Hap2) is a favorable haplotype for increasing GNS, but the thousand-grain weight (TGW) was not significantly different between Haplotype 1 (Hap1) and Hap2.

Wheat (Triticum aestivum L.) is a crucial global food crop that plays a central role in agricultural production and food security. The spike number per unit area (SN) is one of the three component factors of grain yield. In this study, we combined the UG-Map with 27 environments of a recombinant inbred line (RIL) population, and mapped a quantitative trait locus (QTL) for SN, QSn-7A-9048, in which the meta-QTL interval contains only one candidate gene, TraesCS7A02G-364700 (TaKMT-7A). Using the CRISPR/Cas9 system, we generated two homozygous mutant lines, aa-1 and aa-2 of TaKMT-7A, which resulted in frameshift mutations, leading to the premature termination of the translation process. The SN values for the wild type (WT), aa-1, and aa-2 were 4.48, 3.43, and 3.48, respectively. Compared with the WT, the SN of the two mutant lines significantly decreased, and no significant differences for grain number per spike (GNS) and thousand-grain weight (TGW) were detected. We also obtained two overexpression (OE) lines of TaKMT-7A, OE-1 and OE-2. The SN values for the negative control (NC), OE-1, and OE-2 were 2.31, 3.33, and 3.00, respectively. Compared with NC, the SN values in the OE lines significantly increased. The phenotypes of the knockout (KO) lines and OE lines demonstrate that TaKMT-7A acts as a positive regulator of SN in wheat. We performed RNA-Seq analysis using young tiller buds from the WT and aa-1 mutant lines at the tillering stage, and a total of 2315 differentially expressed genes (DEGs) were identified. We screened 22 wheat genes, of which 18 orthologous genes have previously been cloned and are associated with branching in rice and Arabidopsis. These genes included nitrogen transporter, amino metabolism, auxin transporter, auxin homeostasis, auxin response, auxin biosynthesis, strigolactone biosynthesis, and repress gibberellin responses. These genes may represent potential downstream targets of TaKMT-7A.

Wheat (Triticum aestivum L.) is a vital staple crop globally, with its grain microelement content playing a crucial role in human nutrition and health. In this study, the concentrations of eight essential microelements (micronutrients and toxic elements): iron (Fe), manganese (Mn), copper (Cu), zinc (Zn), selenium (Se), chromium (Cr), cadmium (Cd), and arsenic (As), were quantified in 272 wheat varieties using inductively coupled plasma mass spectrometry (ICP-MS) under three different environments. A genome-wide association study (GWAS) was conducted using 176,357 molecular markers, comprising 163,223 single-nucleotide polymorphisms (SNPs) and 13,134 insertion-deletion (InDels) variants, identified through RNA sequencing. A total of 196 significant markers associated with microelement content traits were identified across 21 chromosomes in various environments. Of these, 14 significant markers consistently appeared across environments, forming 13 QTLs and linking to 45 candidate genes. Among these, 29 genes were homologs of known genes in Arabidopsis and rice, while 16 were novel candidates. Haplotype analysis indicated significant phenotypic variation in microelement accumulation, with TraesCS6A02G204300Hap2 notably enhancing iron content. This study provides valuable insights into the genetic architecture of microelement accumulation in wheat grains and introduces novel genetic resources for breeding wheat varieties aimed at improving micronutrient content and ensuring food safety.

In wheat ( Triticum aestivum L.), yield component traits (YCTs) are the most important yield traits. Only several genes for YCTs have been originally cloned. The efficient cloning of genes for YCTs directly from wheat remains a challenge. Here, we proposed a strategy for cloning genes from quantitative trait loci (QTLs) by sequencing of recombinant inbred lines (RILs) (QTL-Seq-RIL). Using the ‘TN18 × LM6’ RIL population as an example, we identified 138 candidate unigenes (CUGs) for YCTs from 77 stable QTLs. The average of CUGs per QTL was 1.8, which enabled us to confirm the CUGs directly. We have confirmed seven CUGs, TaIFABPL, TaDdRp, TaRLK, TaTD, TaTFC3, TaKMT and TaSPL17 , via the CRISPR/Cas9 system. Of these, six genes were found firstly to regulate YCTs in crops except for TaSPL17 . Five CUGs (include TaSPL17 ) for which orthologous genes have been cloned previously with the same or similar agronomic functions. It is to say, 11 CUGs were preliminarily validated using a single RIL population. QTL-Seq-RIL provides an efficient method for rapid gene cloning using existing RIL populations.### Competing Interest StatementThe authors have declared no competing interest.

Fusarium head blight (FHB) stands out as one of the most devastating wheat diseases and leads to significantly grain yield losses and quality reductions in epidemic years. Exploring quantitative trait loci (QTL) for FHB resistance is a critical step for developing new FHB-resistant varieties. We previously constructed a genetic map of unigenes (UG-Map) according to the physical positions using a set of recombinant-inbred lines (RILs) derived from the cross of ‘TN18 × LM6’ (TL-RILs). Here, the number of diseased spikelets (NDS) and relative disease index (RDI) for FHB resistance were investigated under four environments using TL-RILs, which were distributed across 13 chromosomes. A number of 36 candidate genes for NDS and RDI from of 19 stable QTLs were identified. The average number of candidate genes per QTL was 1.89, with 14 (73.7

EARLY FLOWERING 3 (ELF3), a light zeitnehmer (time-taker) gene, regulates circadian rhythm and photoperiodic flowering in Arabidopsis, rice, and barley. The three orthologs of ELF3 (TaELF3-1AL, TaELF3-1BL, and TaELF3-1DL) have been identified in wheat too, and one gene, TaELF3-1DL, has been associated with heading date. However, the basic characteristics of these three genes and the roles of the other two genes, TaELF3-1BL and, TaELF3-1AL, remain unknown. Therefore, the present study obtained the coding sequences of the three orthologs (TaELF3-1AL, TaELF3-1BL, and TaELF3-1DL) of ELF3 from bread wheat and characterized them and investigated the role of TaELF3-1BL in Arabidopsis. Protein sequence comparison revealed similarities among the three TaELF3 genes of wheat; however, they were different from the Arabidopsis ELF3. Real-time quantitative PCR revealed TaELF3 expression in all wheat tissues tested, with the highest expression in young spikes; the three genes showed rhythmic expression patterns also. Furthermore, the overexpression of the TaELF3-1BL gene in Arabidopsis delayed flowering, indicating their importance in flowering. Subsequent overexpression of TaELF3-1BL in the Arabidopsis ELF3 nonfunctional mutant (elf3 mutant) eliminated its early flowering phenotype, and slightly delayed flowering. The wild-type Arabidopsis overexpressing TaELF3-1BL demonstrated reduced expression levels of flowering-related genes, such as CONSTANS (AtCO), FLOWERING LOCUS T (AtFT), and GIGANTEA (AtGI). Thus, the study characterized the three TaELF3 genes and associated TaELF3-1BL with flowering in Arabidopsis, suggesting a role in regulating flowering in wheat too. These findings provide a basis for further research on TaELF3 functions in wheat.

小麦黄花叶病是我国黄淮麦区一种重要的小麦病害.本研究对134份品种(系)进行小麦黄花叶病抗性鉴定;结合90K SNP芯片基因型分型数据,对小麦黄花叶病病情指数进行全基因组关联分析.结果表明,134份供试材料中,27个品种(系)对小麦黄花叶病表现为高抗,占供试材料的20.15％;21个品种(系)表现中抗,占供试材料的15.67％.通过全基因组关联分析(GWAS),检测到56个SNP标记与小麦黄花叶病病情指数显著关联,分别位于2A、2B、2D、3A、4B、7B染色体上,可解释8.8％～29.6％的表型变异.其中,在2D染色体上检测到在3个环境及其最佳线性无偏预测值(BLUP)中均显著关联的SNP标记有40个,位于76.57～86.28 cM区间.