Cloning and Development of Molecular Marker for the Wheat (Triticum aestivum) Cell Wall Invertase Gene TaCWINV41
ZHANG Yu-Xuan1,2,*, CHU Yu-Hang1,2,*, XUE Bo-Yang1,2, MENG Heng-Yu1,2, ZHU Xue-Yuan1,2, WANG Guang-Liang1,2, LIU Bing-Liang1,2, ZHANG Rong-Zhi3, REN Yan4,5,**, LIU Jin-Dong6,**, FU Lu-Ping1,2,**
1 Key Laboratory of Crop Genomics and Molecular Breeding of Jiangsu Province/Key Laboratory of Plant Functional Genomics of the Ministry of Education/Key Laboratory of Crop Genetics and Physiology of Jiangsu Province/Agricultural College, Yangzhou University, Yangzhou 225009, China; 2 Yangzhou University/Collaborative Innovation Center for Modern Industrial Technology of Grain Crops of Jiangsu Province, Yangzhou 225009, China; 3 Crop Research Institute, Shandong Academy of Agricultural Sciences, Jinan 250100, China; 4 State Key Laboratory of High-Efficiency Production of Wheat-Maize Double Cropping, Beijing 100193, China; 5 College of Agronomy, Henan Agricultural University, Zhengzhou 450046, China; 6 Institute of Crop Science, Chinese Academy of Agriculture Sciences, Beijing 100081, China
Abstract：Grain filling rate (GFR) is a key factor influencing wheat (Triticum aestivum) thousand-kernel weight (TKW) and yield, while cell wall invertases (CWINVs) are core enzymes regulating plant carbohydrate metabolism and grain filling. It is of high significance to explore variations of wheat CWINV family genes and develop molecular markers for genetic improvement of wheat GFR and yield. In this study, a natural population comprising 166 wheat varieties (advanced lines) from the Yellow and Huai Valleys Winter Wheat Zone was used to measure the maximum grain filling rate (GFRmax) and TKW under 4 environments. With the genotyping data of wheat 660K and 90K SNP chips, genome-wide association study (GWAS) was conducted and a major-effect QTL simultaneously associated with GFRmax and TKW was identified at the 60.60~62.33 Mb interval on chromosome 4B, explaining 7.48%~12.40% and 7.53%~9.50% of the phenotypic variance for GFRmax and TKW, respectively. Referencing the 'Chinese Spring' reference genome, the TaCWINV41 gene (TraesCS4B02G066000) belonging to the CWINV family was located within the interval of this QTL. TaCWINV41 gene was cloned from cultivars 'Yangmai 16' and 'Zhongmai 895', and 2 SNPs were identified in its third exon; one of which, at position 1 892, was a C/T missense mutation. Based on this SNP, a kompetitive allele-specific PCR (KASP) marker, TaCWINV41_KASP, was developed. The reliability of this marker was validated using GFRmax and TKW phenotypic data of 194 wheat varieties. t-test analysis revealed that varieties with TT genotype exhibited significantly higher GFRmax and TKW than those with CC genotype (P<0.05, P<0.01, or P<0.001), confirming the value of TaCWINV41_KASP for molecular breeding. In summary, this study identified a major QTL simultaneously regulating wheat GFR and TKW through GWAS, uncovered allelic variation in the candidate gene TaCWINV41 at this locus, and developed a KASP marker for molecular breeding of wheat GFR and TKW. These findings provide theoretical insights into the genetic regulation of wheat GFR and offer an efficient molecular tool for genetic improvement of GFR and grain yield.
[1] 高翔, 董剑, 庞红喜. 2002. 小麦高产品种籽粒灌浆与粒重的关系[J]. 西北农业学报, 11(3): 33-35. (Gao X, Dong J, Pang H X.2002. Relationship of grain-filling and grain weight in high-yield wheat varieties[J]. Acta Agriculturae Boreali-occidentalis Sinica. 11(3): 33-35.) [2] 郭艳艳, 段巍巍. 2018. 不同冬小麦品种籽粒胚乳增殖和灌浆对粒重的影响[J]. 麦类作物学报, 38(1): 84-89. (Guo Y Y, Duan W W.2018. Studies on endosperm development and grain filling characteristics in different winter wheat varieties[J]. Journal of Triticeae Crops, 38(1): 84-89.) [3] 雷钰欣, 曹子千, 李纪璇, 等. 2024. 高等植物转化酶的研究进展[J]. 分子植物育种, 22(4): 1242-1250. (Lei Y X, Cao Z Q, Li J X, et al.2024. Research progress of invertase in higher plants[J]. Molecular Plant Breeding, 22(4): 1242-1250.) [4] 刘大同, 余徐润, 朱冬梅, 等. 2017. 扬麦16 “灌浆快”与颖果显微结构和内源生长素的关系[J]. 麦类作物学报, 37(6): 739-749. (Liu D T, Yu X R, Zhu D M, et al.2017. Relationship between caryopsis microstructure and endogenous auxin level and the fast grain filling of Yangmai 16[J]. Journal of Triticeae Crops, 37(6): 739-749.) [5] 刘源霞, 王文文, 张保望, 等. 2014. 小麦籽粒灌浆特性与千粒重的相关性分析[J]. 东北农业大学学报, 45(4): 12-17. (Liu Y X, Wang W W, Zhang B W, et al.2014. Correlation analysis of grain filling characteristics and thousand-grain weight in wheat[J]. Journal of Northeast Agricultural University, 45(4): 12-17.) [6] 苗永杰, 阎俊, 赵德辉, 等. 2018. 黄淮麦区小麦主栽品种粒重与籽粒灌浆特性的关系[J]. 作物学报, 44(2): 260-267. (Miao Y J, Yan J, Zhao D H, et al.2018. Relationship between grain filling parameters and grain weight in leading wheat cultivars in the Yellow and Huai Rivers Valley[J]. Aata Agronomica Sinica, 44(2): 260-267.) [7] 孙晓晓. 2018. 小麦籽粒灌浆速率的基因定位及粒重分子标记开发[D]. 硕士学位论文, 山东农业大学, 导师: 陈建省, pp. 30-31. (Sun X X.2018. QTL mapping for grain filling rate in wheat (Triticum aestivum L.) and molecular markers development for thousand kernel weight[D]. Thesis for M.S., Shandong Agricultural University, Supervisor: Chen J S, pp. 30-31.) [8] 孙炎. 2025. 小麦籽粒灌浆速率全基因组关联分析[D]. 硕士学位论文, 扬州大学, 导师: 付路平, pp. 12-40. (Sun Y.2025. Genome-wide association study on grain filling rate in wheat[D]. Thesis for M.S., Yangzhou University, Supervisor: Fu L P, pp. 12-40.) [9] 田小萍, 王光浩, 张宏, 等. 2020. 小麦灌浆相关基因TaGIF1的克隆及表达分析[J]. 麦类作物学报, 40(1): 30-40. (Tian X P, Wang G H, Zhang H, et al.2020. Cloning and expression analysis of TaGIF1 gene related to grain filling in common wheat[J]. Journal of Triticeae Crops, 40(1): 30-40.) [10] 王珂, 杨娜, 席吉龙, 等. 2022. 三种数学模型模拟不同播期小麦籽粒灌浆过程的比较分析[J]. 麦类作物学报, 42: 1398-1407. (Wang K, Yang N, Xi J L, et al.2022. Comparison of three mathematical equation for simulating the wheat grain filling process with different sowing dates[J]. Journal of Triticeae Crops, 42: 1398-1407.) [11] 王瑞霞, 张秀英, 伍玲, 等. 2008. 不同生态环境条件下小麦籽粒灌浆速率及千粒重QTL分析[J]. 作物学报, 34(10): 1750-1756. (Wang R X, Zhang X Y, Wu L, et al.2008. QTL mapping for grain filling rate and thousand-grain weight in different ecological environments in wheat[J]. Aata Agronomica Sinica, 34(10): 1750-1756.) [12] 左西龙, 王正波, 陈春生, 等. 2025. 不同小麦品种籽粒灌浆特性及产量品质研究[J]. 安徽农业科学, 53(9): 34-39. (Zuo X L, Wang Z B, Chen C S, et al.2025. Study on grain filling characteristics, yield and quality of different wheat varieties[J]. Journal of Anhui Agricultural Sciences, 53(9): 34-39.) [13] Alberto F, Bignon C, Sulzenbacher G, et al.2004. The three-dimensional structure of invertase (β-fructosidase) from Thermotoga maritima reveals a bimodular arrangement and an evolutionary relationship between retaining and inverting glycosidases[J]. Journal of Biological Chemistry, 279(18): 18903-18910. [14] Bradbury P J, Zhang Z, Kroon D E, et al.2007. TASSEL: Software for association mapping of complex traits in diverse samples[J]. Bioinformatics, 23(9): 2633-2635. [15] Cho J I, Lee S K, Ko S H, et al.2005. Molecular cloning and expression analysis of the cell-wall invertase gene family in rice (Oryza sativa L.)[J]. Plant Cell Reports, 24(4): 225-236. [16] Fu L P, Wu J C, Yang S R, et al.2020. Genome-wide association analysis of stem water-soluble carbohydrate content in bread wheat[J]. Theoretical and Applied Genetics, 133(10): 2897-2914. [17] Ishimaru T, Hirose T, Matsuda T, et al.2005. Expression patterns of genes encoding carbohydrate-metabolizing enzymes and their relationship to grain filling in rice (Oryza sativa L.): Comparison of caryopses located at different positions in a panicle[J]. Plant and Cell Physiology, 46: 620-628. [18] IWGSC.2018. Shifting the limits in wheat research and breeding using a fully annotated reference genome[J]. Science, 361: eaar7191. [19] Karuppiah N, Vadlamudi B, Kaufman P B.1989. Purification and characterization of soluble (cytosolic) and bound (cell wall) isoforms of invertases in barley (Hordeum vulgare) elongating stem tissue[J]. Plant Physiology, 91(3): 993-998. [20] Kim D, Lee G, Chang M, et al.2011. Purification and biochemical characterization of insoluble acid invertase (INAC-INV) from pea seedlings[J]. Journal of Agricultural and Food Chemistry, 59(20): 11228-11233. [21] Lee S K, Shim S H, Eom J S, et al.2024. Cell wall invertases from maternal tissues modulate sucrose flux in apoplastic pathways during rice anther and seed development[J]. International Journal of Molecular Science, 25(21): 11557. [22] Lin Y, Jiang X, Tao Y, et al.2020. Identification and validation of stable quantitative trait loci for grain filling rate in common wheat (Triticum aestivum L.)[J]. Theoretical and Applied Genetics, 133(8): 2377-2385. [23] Liu J D, He Z H, Rasheed A, et al.2017. Genome-wide association mapping of black point reaction in common wheat (Triticum aestivum L.)[J]. BMC Plant Biology, 17(1): 220. [24] Sturm A, Tang G Q.1999. The sucrose-cleaving enzymes of plants are crucial for development, growth and carbon partitioning[J]. Trends in Plant Science, 4(10): 401-407. [25] Verhaest M, Lammens W, Le Roy K, et al.2006. X-ray diffraction structure of a cell-wall invertase from Arabidopsis thaliana[J]. Acta Crystallographica Section D-Structural Biology, 62(12): 1555-1563. [26] Wang E, Wang J, Zhu X D, et al.2008. Control of rice grain-filling and yield by a gene with a potential signature of domestication[J]. Nature Genetics, 40(11): 1370-1374. [27] Ye J, Bu Y, He M, et al.2022. Genome-wide analysis of invertase gene family in wheat (Triticum aestivum L.) indicates involvement of TaCWINVs in pollen development[J]. Plant Growth Regulation, 98: 77-89.
