Expression studies of the candidate genes

Apical and basal spikelets were sampled on 6, 9, and 12 days after anthesis in both parents PDK Shriram and Heera, frozen into liquid nitrogen, and stored at – 80 °C until use. Among RILs, one high spikelet number compact panicle line, 166A, and one lax panicle low spikelet number line, 14A were also considered for the sampling of the spikelets, similar to that of the parents. Total RNA was isolated from the collected samples using TRIZOL reagent (Invitrogen) following the reagent user manual. The quality and quantity of the RNA isolated from the individual samples were checked on Agarose gel and Nanodrop spectrophotometer, respectively. Quanti-Tect Reverse Transcription Kit (Qiagen) was used for conversion of total isolated RNA to cDNA following the protocol outlined in the kit’s manual. The study of the expression of some genes associated with identified QTLs regions were conducted using the cDNAs prepared from the individual samples. Expression of a gene was studied by qRT-PCR taking the cDNA as template and SYBR green (Agilent). Primers specific to the gene of interest were designed using Primer Blast software at the NCBI site. The required amount of SYBR green, cDNA template, and the primers for a gene was mixed in a final volume of 20 µl, and PCR was run in Real-Time PCR (Bio-Rad CFX). Rice actin was used as an internal control. The relative level of templates of the individual gene in the apical and basal spikelets was quantified following Pfaffl⁶⁶, and the result was expressed as a fold change in expression in the basal spikelets over apical ones. Seven candidate genes were selected from three QTL regions and used in the expression studies (Supplementary Table ^S6).