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Journal of Integrative Agriculture
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TaRLK-4B, a novel gene regulating grain number per spike in wheat

Hui Wang1*, Mingxia Zhang2*, Junsheng Sun3, Haimeng Wu1, Yijun Wang1, Huiyan Xu1, Jianwei Sun1, Guangde Lv4, Baojin Guo5, Xuemei Jin6, Rugang Wu7, Ying Guo1, Yanrong An1#, Yan Zhao1#, Sishen Li1#

1 State Key Laboratory of Wheat Improvement, College of Agronomy, Shandong Agricultural University, Tai’an 271018, China

2 Jinan Academy of Agricultural Science, Jinan 250316, China

3 Department of Biological and Chemical Engineering, Jining Polytechnic, Jining 272004, China

4 Tai’an Academy of Agricultural Science, Tai’an 271000, China

5 State Key Laboratory of Crop Gene Resources and Breeding, Key Laboratory of Grain Crop Genetic Resources Evaluation and Utilization, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China

6 Rizhao Academy of Agricultural Sciences, Rizhao 276800, China

7 Dezhou Academy of Agricultural Sciences, Dezhou 253000, China

 Highlights 

l   A novel gene, TraesCS4B02G047100 (TaRLK-4B), was identified for grain number per spike (GNS) using both quantitative trait locus (QTL) mapping and genome-wide association study (GWAS) based on RNA-Seq.

l   The positive regulation of GNS, total spikelet number per spike (TSS) and spike length (SL) by TaRLK-4B was validated using the CRISPR/Cas9 gene editing system.

l   Combining the RNA-Seq and DAP-Seq data, we hypothesize that TaRLK-4B functions within a regulatory network centered on TaSPL17 to modulate GNS and associated agronomic traits.

l   Haplotype 2 (Hap2) significantly increased GNS and was a favorable haplotype.

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摘要  

小麦是全球最重要的粮食作物之一,穗粒数(GNS)是决定小麦产量的关键性状之一。因此,鉴定调控穗粒数基因对小麦生产具有重要意义。但小麦为异源六倍体作物,基因组高度复杂,基因分离难度较大。本研究通过数量性状位点(QTL)定位与基于转录组测序(RNA-Seq)的全基因组关联分析(GWAS),鉴定出一个调控穗粒数的候选基因TraesCS4B02G047100TaRLK-4B)。利用CRISPR/Cas9基因编辑技术获得bb-1bb-2bb-3三个纯合突变株系。与野生型Fielder(WT)相比,三个突变株系的穗粒数、总小穗数(TSS)及穗长(SL)均显著降低。该结果表明,TaRLK-4B对穗粒数、总小穗数与穗长具有正向调控作用。TaRLK-4B基因包含2个外显子与1个内含子,属于类受体激酶家族中最大的LRR-RLK亚家族。本研究对分蘖期WT与bb-2的幼穗开展RNA-Seq分析,共鉴定出1193个差异表达基因(DEGs),其中包含小麦三个亚基因组中已报道的TaSPL17同源基因,及其水稻直系同源基因OsIPA1OsSPL14)。通过整合TaRLK-4B的RNA-Seq数据与TaSPL17-7D的DAP-Seq数据,本研究筛选获得136个重叠基因,推测其为TaSPL17-7D的潜在下游靶标基因。据此推测TaRLK-4BTaSPL17核心调控网络中一个未被报道的新组分。此外,单倍型分析结果表明,单倍型2(Hap2)为提升穗粒数的优异单倍型。而单倍型1(Hap1)与Hap2的千粒重(TGW)无显著差异。



Abstract  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.
Keywords:  wheat       grain number per spike (GNS)              quantitative trait locus (QTL)              genome-wide association study (GWAS)              TraesCS4B02G047100              receptor-like kinase (RLK)              CRISPR/Cas9 system  
Online: 30 May 2026  
Fund: 

This research was supported by the Taishan Scholars Program of Shandong Province, China(tsqn202408131)and the Modern Agricultural Industry Technology System of Shandong Province, China (SDAIT-01-04).

About author:  #Correspondence Sishen Li, E-mail: ssli@sdau.edu.cn; Yan Zhao, E-mail: zhaoyan@sdau.edu.cn; Yanrong An, E-mail: anyr@sdau.edu.cn * Indicates the authors who contributed equally to this study.

Cite this article: 

Hui Wang, Mingxia Zhang, Junsheng Sun, Haimeng Wu, Yijun Wang, Huiyan Xu, Jianwei Sun, Guangde Lv, Baojin Guo, Xuemei Jin, Rugang Wu, Ying Guo, Yanrong An, Yan Zhao, Sishen Li. 2026. TaRLK-4B, a novel gene regulating grain number per spike in wheat. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2026.05.040

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