Identification and functional analysis of a chromosome 2D fragment harboring TaFPF1 gene with the potential for yield improvement using a late heading wheat mutant.

KEY MESSAGE: A chromosome fragment influencing wheat heading and grain size was identified using mapping of m406 mutant. The study of TaFPF1 in this fragment provides more insights into wheat yield improvement. In recent years, wheat production has faced formidable challenges driven by rapid population growth and climate change, emphasizing the importance of improving specific agronomic traits such as heading date, spike length, and grain size. To identify potential genes for improving these traits, we screened a wheat EMS mutant library and identified a mutant, designated m406, which exhibited a significantly delayed heading date compared to the wild-type. Intriguingly, the mutant also displayed significantly longer spike and larger grain size. Genetic analysis revealed that a single recessive gene was responsible for the delayed heading. Surprisingly, a large 46.58 Mb deletion at the terminal region of chromosome arm 2DS in the mutant was identified through fine mapping and fluorescence in situ hybridization. Thus, the phenotypes of the mutant m406 are controlled by a group of linked genes. This deletion encompassed 917 annotated high-confidence genes, including the previously studied wheat genes Ppd1 and TaDA1, which could affect heading date and grain size. Multiple genes in this region probably contribute to the phenotypes of m406. We further investigated the function of TaFPF1 using gene editing. TaFPF1 knockout mutants showed delayed heading and increased grain size. Moreover, we identified the direct upstream gene of TaFPF1 and investigated its relationship with other important flowering genes. Our study not only identified more genes affecting heading and grain development within this deleted region but also highlighted the potential of combining these genes for improvement of wheat traits.

Mutagenesis-Derived Resistance to Black Point in Wheat.

Black point, a severe global wheat disease, necessitates deploying resistant cultivars for effective control. However, susceptibility remains prevalent among most wheat cultivars. Identifying new sources of resistance and understanding their mechanisms are crucial for breeding resistant cultivars. This study pinpointed black point resistance in an ethyl methane sulfonate (EMS)-mutagenized wheat population of Wanyuanbai 1 (WYB) and analyzed resistant mutants using RNA-Seq. The findings revealed the following: (i) wyb-18, among 10,008 EMS-mutagenized lines, exhibited robust resistance with significantly lower black point incidence under artificial Bipolaris sorokiniana inoculation in 2020 and 2021 (average incidence of 5.2% over 2 years), markedly reduced compared with WYB (50.9%). (ii) wyb-18 kernels displayed black point symptoms at 12 days after inoculation (dai), 3 days later than WYB. At 15 dai, wyb-18 kernels had isolated black spots, unlike WYB kernels, where the entire embryo turned black. (iii) wyb-18 showed heightened antioxidant enzyme activity, including peroxidase, catalase, and superoxide dismutase. (iv) Analysis of 543 differentially expressed genes between wyb-18 and WYB at 9 dai identified enrichment in the MAPK signaling pathway through KEGG analysis. Ten genes in this pathway exhibited upregulated expression, while one was downregulated in wyb-18. Among these genes, PR1, WRKY11, SAPK5, and TraesCS1A02G326800 (chitin recognition protein) consistently showed upregulation in wyb-18, making them potential candidates for black point resistance. These results offer valuable germplasm resources for breeding and novel insights into the mechanisms of black point resistance.

Screening and Resistance Locus Identification of the Mutant fcrZ22 Resistant to Crown Rot Caused by Fusarium pseudograminearum.

Crown rot caused by Fusarium pseudograminearum is a devastating wheat disease worldwide. In addition to yield losses, the fungi causing Fusarium crown rot (FCR) also deteriorate the quality and safety of food because of the production of mycotoxins. Planting resistant cultivars is an effective way to control FCR. However, most wheat cultivars are susceptible to FCR. Therefore, development of new sources and detection of loci for FCR resistance are necessary. In the present study, a resistant mutant, fcrZ22, was identified from an ethyl methane sulfonate (EMS)-mutagenized population of the cultivar Zhoumai 22, and then fcrZ22 was crossed with the wild type to produce an F2 population. Genetic analysis of the F2 population was carried out by the mixed inheritance model of major genes plus polygenes, and 20 resistant and 20 susceptible plants were selected to assemble mixed pools. Combining 660K SNP arrays, the resistance loci were detected by bulked segregant analysis. The resistance to FCR caused by F. pseudograminearum in the F2 population was in accordance with the "mixed model with two major genes of additive-epistasis effect + additive-dominant polygenes," and the heritability of the major gene was 0.92. Twenty-one loci were detected, which were located on 10 chromosomes, namely, 1B (1), 1D (1), 2A (3), 1B (1), 3A (3), 3B (3), 4A (2), 5A (2), 7A (3), and 7B (2). Among the 21 loci, eight were new loci for FCR resistance. This is the first report of detecting loci for FCR resistance from a mutant. The results of the present study provided excellent germplasm resources for breeding wheat cultivars with FCR resistance and laid the foundation for fine mapping of FCR resistance loci.

Identification of the early leaf senescence gene ELS3 in bread wheat (Triticum aestivum L.).

MAIN CONCLUSION: Characterization of the early leaf senescence mutant els3 and identification of its causal gene ELS3, which encodes an LRR-RLK protein in wheat. Leaf senescence is an important agronomic trait that affects both crop yield and quality. However, few senescence-related genes in wheat have been cloned and functionally analyzed. Here, we report the characterization of the early leaf senescence mutant els3 and fine mapping of its causal gene ELS3 in wheat. Compared with wild-type Yanzhan4110 (YZ4110), the els3 mutant had a decreased chlorophyll content and a degraded chloroplast structure after the flowering stage. Further biochemical assays in flag leaves showed that the superoxide anion and hydrogen peroxide contents increased, while the activities of antioxidant enzymes, including catalase, superoxide dismutase and glutathione reductase, decreased gradually after the flowering stage in the els3 mutant. To clone the causal gene underlying the phenotype of leaf senescence, a genetic map was constructed using 10,133 individuals of F2:3 populations, and ELS3 was located in a 2.52 Mb region on chromosome 2DL containing 16 putative genes. Subsequent sequence analysis and gene annotation identified only one SNP (C to T) in the first exon of TraesCS2D02G332700, resulting in an amino acid substitution (Pro329Ser), and TraesCS2D02G332700 was preliminarily considered as the candidate gene of ELS3. ELS3 encodes a leucine-rich repeat receptor-like kinase (LRR-RLK) protein that is localized on the cell membrane. We also found that the transient expression of mutant TraesCS2D02G332700 can induce leaf senescence in N. benthamiana. Taken together, TraesCS2D02G332700 is likely to be the candidate gene of ELS3 and may have a function in regulating leaf senescence.

A Glu209Lys substitution in DRG1/TaACT7, which disturbs F-actin organization, reduces plant height and grain length in bread wheat.

Plant height and grain size are two important agronomic traits that are closely related to crop yield. Numerous dwarf and grain-shape mutants have been studied to identify genes that can be used to increase crop yield and improve breeding programs. In this study, we characterized a dominant mutant, dwarf and round grain 1 (drg1-D), in bread wheat (Triticum aestivum L.). drg1-D plants exhibit multiple phenotypic changes, including dwarfism, round grains, and insensitivity to brassinosteroids (BR). Cell structure observation in drg1-D mutant plants showed that the reduced organ size is due to irregular cell shape. Using map-based cloning and verification in transgenic plants, we found that a Glu209Lys substitution in the DRG1 protein is responsible for the irregular cell size and arrangement in the drg1-D mutant. DRG1/TaACT7 encodes an actin family protein that is essential for polymerization stability and microfilament (MF) formation. In addition, the BR response and vesicular transport were altered by the abnormal actin cytoskeleton in drg1-D mutant plants. Our study demonstrates that DRG1/TaACT7 plays an important role in wheat cell shape determination by modulating actin organization and intracellular material transport, which could in the longer term provide tools to better understand the polymerization of actin and its assembly into filaments and arrays.

Tiller Number1 encodes an ankyrin repeat protein that controls tillering in bread wheat.

Wheat (Triticum aestivum L.) is a major staple food for more than one-third of the world's population. Tiller number is an important agronomic trait in wheat, but only few related genes have been cloned. Here, we isolate a wheat mutant, tiller number1 (tn1), with much fewer tillers. We clone the TN1 gene via map-based cloning: TN1 encodes an ankyrin repeat protein with a transmembrane domain (ANK-TM). We show that a single amino acid substitution in the third conserved ankyrin repeat domain causes the decreased tiller number of tn1 mutant plants. Resequencing and haplotype analysis indicate that TN1 is conserved in wheat landraces and modern cultivars. Further, we reveal that the expression level of the abscisic acid (ABA) biosynthetic gene TaNCED3 and ABA content are significantly increased in the shoot base and tiller bud of the tn1 mutants; TN1 but not tn1 could inhibit the binding of TaPYL to TaPP2C via direct interaction with TaPYL. Taken together, we clone a key wheat tiller number regulatory gene TN1, which promotes tiller bud outgrowth probably through inhibiting ABA biosynthesis and signaling.

Combining a New Exome Capture Panel With an Effective varBScore Algorithm Accelerates BSA-Based Gene Cloning in Wheat.

The discovery of functional genes underlying agronomic traits is of great importance for wheat improvement. Here we designed a new wheat exome capture probe panel based on IWGSC RefSeq v1.0 genome sequence information and developed an effective algorithm, varBScore, that can sufficiently reduce the background noise in gene mapping and identification. An effective method, termed bulked segregant exome capture sequencing (BSE-Seq) for identifying causal mutations or candidate genes was established by combining the use of a newly designed wheat exome capture panel, sequencing of bulked segregant pools from segregating populations, and the robust algorithm varBScore. We evaluated the effectiveness of varBScore on SNP calling using the published dataset for mapping and cloning the yellow rust resistance gene Yr7 in wheat. Furthermore, using BSE-Seq, we rapidly identified a wheat yellow leaf mutant gene, ygl1, in an ethyl methanesulfonate (EMS) mutant population and found that a single mutation of G to A at 921 position in the wild type YGL1 gene encoding magnesium-chelatase subunit chlI caused the leaf yellowing phenotype. We further showed that mutation of YGL1 through CRISPR/Cas9 gene editing led to a yellow phenotype on the leaves of transgenic wheat, indicating that ygl1 is the correct causal gene responsible for the mutant phenotype. In summary, our approach is highly efficient for discovering causal mutations and gene cloning in wheat.

Transcriptome Analysis of a Premature Leaf Senescence Mutant of Common Wheat (Triticum aestivum L.).

Leaf senescence is an important agronomic trait that affects both crop yield and quality. In this study, we characterized a premature leaf senescence mutant of wheat (Triticum aestivum L.) obtained by ethylmethane sulfonate (EMS) mutagenesis, named m68. Genetic analysis showed that the leaf senescence phenotype of m68 is controlled by a single recessive nuclear gene. We compared the transcriptome of wheat leaves between the wild type (WT) and the m68 mutant at four time points. Differentially expressed gene (DEG) analysis revealed many genes that were closely related to senescence genes. Gene Ontology (GO) enrichment analysis suggested that transcription factors and protein transport genes might function in the beginning of leaf senescence, while genes that were associated with chlorophyll and carbon metabolism might function in the later stage. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the genes that are involved in plant hormone signal transduction were significantly enriched. Through expression pattern clustering of DEGs, we identified 1012 genes that were induced during senescence, and we found that the WRKY family and zinc finger transcription factors might be more important than other transcription factors in the early stage of leaf senescence. These results will not only support further gene cloning and functional analysis of m68, but also facilitate the study of leaf senescence in wheat.

The wheat MYB transcription factor TaMYB18 regulates leaf rolling in rice.

Leaf rolling is an important agronomic trait in crop breeding. Moderate leaf rolling maintains the erectness of leaves and minimizes shadowing between leaves, leading to improved photosynthetic efficiency. Although some genes controlling leaf rolling have been isolated from rice and other plant species, few studies have examined leaf rolling in wheat. In the present study, the leaf rolling regulator gene, TaMYB18, was identified in a large-scale transgene project involving the transformation of 1455 wheat transcription factor genes into rice. Three homologous sequences of TaMYB18 were isolated from hexaploid wheat and localized to chromosomes 5A, 5B and 5D, respectively. TaMYB18, an R2R3-MYB transcription factor, localized to the nucleus. TaMYB18 overexpression induced leaf rolling in transgenic rice. Additionally, the three members of TaMYB18 exhibited functional redundancy in rice. Furthermore, the function of TaMYB18 in regulating leaf rolling in rice was a dose-dependent. Taken together, these results indicate that TaMYB18 may play an important role in the regulation of leaf development.

The wheat MYB-related transcription factor TaMYB72 promotes flowering in rice.

Through large-scale transformation analyses, TaMYB72 was identified as a flowering time regulator in wheat. TaMYB72 is a MYB family transcription factor localized to the nucleus. Three TaMYB72 homologs, TaMYB72-A, TaMYB72-B and TaMYB72-D, cloned from hexaploid wheat were mapped to the short arm of the group 6 chromosomes. Under the long-day conditions, over-expression of the TaMYB72 in rice shortened the flowering time by approximately 12 d. Expression analyses suggest that TaMYB72 may function through up-regulation of florigen genes Hd3a and RFT1.