Plants 2022, 11, 2288. https://doi.org/10.3390/plants11172288 www.mdpi.com/journal/plants Article Mapping and Validation of qHD7b: Major Heading‐Date QTL Functions Mainly Under Long‐Day Conditions Amir Sohail 1 , Liaqat Shah 1,2 , Ling Liu 1 , Anowerul Islam 1,3 , Zhengfu Yang 1,4 , Qinqin Yang 1 , Galal Bakr Anis 1,5 , Peng Xu 1 , Riaz Muhammad Khan 1,6 , Jiaxin Li 1 , Xihong Shen 1 , Shihua Cheng 1 , Liyong Cao 1,7 , Yingxin Zhang 1, * and Weixun Wu 1, * 1 State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China 2 Department of Agriculture, Mir Chakar Khan Rind University, Sibi 82000, Pakistan 3 Department of Agricultural Extension, Ministry of Agriculture, Dhaka 1215, Bangladesh 4 State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou 311300, China 5 Rice Research and Training Center, Field Crops Research Institute, Agriculture Research Center, Kafrelsheikh 33717, Egypt 6 Cereal Crops Research Institute (CCRI) Pirsabak Nowshera, Agriculture Research System Khyber Pakhtunkhwa 24100, Pakistan 7 Northern Center of China National Rice Research Institute, Shuangyashan 155600, China * Correspondence: wuweixun@caas.cn (W.W.); zhangyingxin@caas.cn (Y.Z.) Abstract: Heading date (HD) is one of the agronomic traits that influence maturity, regional adaptability, and grain yield. The present study was a follow‐up of a previous quantitative trait loci (QTL) mapping study conducted on three populations, which uncovered a total of 62 QTLs associated with 10 agronomic traits. Two of the QTLs for HD on chromosome 7 (qHD7a and qHD7b) had a common flanking marker (RM3670) that may be due to tight linkage, and/or weakness of the statistical method. The objectives of the present study were to map QTLs associated with HD in a set of 76 chromosome segment substitution lines (CSSLs), fine map and validate one of the QTLs (qHD7b) using 2,997 BC5F2:3 plants, and identify candidate genes using sequencing and expression analysis. Using the CSSLs genotyped with 120 markers and evaluated under two short‐day and two long‐day growing conditions, we uncovered a total of fourteen QTLs (qHD2a, qHD4a, qHD4b, qHD5a, qHD6a, qHD6b, qHD7b, qHD7c, qHD8a, qHD10a, qHD10b, qHD11a, qHD12a, and qHD12b). However, only qHD6a and qHD7b were consistently detected in all four environments. The phenotypic variance explained by qHD6a and qHD7b varied from 10.1% to 36.1% (mean 23.1%) and from 8.1% to 32.8% (mean 20.5%), respectively. One of the CSSL lines (CSSL52), which harbored a segment from the early heading XieqingzaoB (XQZB) parent at the qHD7b locus, was then used to develop a BC5F2:3 population for fine mapping and validation. Using a backcross population evaluated for four seasons under different day lengths and temperatures, the qHD7b interval was delimited to a 912.7‐kb region, which is located between RM5436 and RM5499. Sequencing and expression analysis revealed a total of 29 candidate genes, of which Ghd7 (Os07g0261200) is a well‐ known gene that affects heading date, plant height, and grain yield in rice. The ghd7 mutants generated through CRISPR/Cas9 gene editing exhibited early heading. Taken together, the results from both the previous and present study revealed a consistent QTL for heading date on chromosome 7, which coincided not only with the physical position of a known gene, but also with two major effect QTLs that controlled the stigma exertion rate and the number of spikelets in rice. The results provide contributions to the broader adaptability of marker‐assisted breeding to develop high‐yield rice varieties. Keywords: rice (Oryza sativa L.); quantitative trait locus; chromosome segment substitution lines; qHD7b; fine‐mapping Citation: Sohail, A.; Shah, L.; Liu, L.; Islam, A.; Yang, Z.; Yang, Q.; Anis, G.B.; Xu, P.; Khan, R.M.; Li, J.; et al. Mapping and Validation of qHD7b: Major Heading‐Date QTL Functions Mainly Under Long‐Day Conditions. Plants 2022, 11, 2288. https://doi.org/ 10.3390/plants11172288 Academic Editors: Kassa Semagn and Tika Adhikari Received: 28 June 2022 Accepted: 30 August 2022 Published: 1 September 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/license s/by/4.0/).