Molecular-assisted breeding is an effective way to improve targeted agronomic traits. dep1 (dense and erect panicle 1) is a pleiotropic gene that regulates yield, quality, disease resistance, and stress tolerance, traits that are of great value in rice (Oryza sativa L.) breeding. In this study, a colorimetric LAMP (loop-mediated isothermal amplification) assay was developed for the detection of the dep1 allele and tested for the screening and selection of the heavy-panicle hybrid rice elite restorer line SHUHUI498, modified with the allele. InDel (Insertion and Deletion) primers (DEP1_F and DEP1_R) and LAMP primers (F3, B3, FIP, and BIP) for genotyping were designed using the Primer3 Plus (version 3.3.0) and PrimerExplore (version 5) software. Our results showed that both InDel and LAMP markers could be used for accurate genotyping. After incubation at a constant temperature of 65 °C for 60 min with hydroxynaphthol blue (HNB) as a color indicator, the color of the LAMP assay containing the dep1 allele changed to sky blue. The SHUHUI498 rice line that was detected in our LAMP assay displayed phenotypes consistent with the dep1 allele such as having a more compact plant architecture, straight stems and leaves, and a significant increase in the number of effective panicles and spikelets, demonstrating the effectiveness of our method in screening for the dep1 allele in rice breeding.

The GROWTH-REGULATING FACTOR4 (OsGRF4) allele is an important target for the development of new high nitrogen-use efficiency (NUE) rice lines that would require less fertilizers. Detection of OsGRF4 through PCR (polymerase chain reaction)-based assay is cumbersome and needs advanced laboratory skills and facilities. Hence, a method for conveniently and rapidly detecting OsGRF4 on-field is a key requirement for further research and applications. In this study, we employed cleaved amplified polymorphic sequences (CAPs) and loop-mediated isothermal amplification (LAMP) techniques to develop a convenient visual detection method for high NUE gene OsGRF4^NM73 (OsGRF4 from the rice line NM73). The TC→AA mutation at 1187–1188 bp loci was selected as the target sequence for the OsGRF4^NM73 allele. We further employed this method of identification in 10 rice varieties that carried the OsGRF4 gene and results revealed that one variety (NM73) carries the target OsGRF4^NM73 allele, while other varieties did not possess the osgrf4 genotype. The optimal LAMP reaction using hydroxynaphthol blue (HNB), a chromogenic indicator, was carried out at 65 °C for 60 min, and the presence of OsGRF4^NM73 allele was confirmed by color changes from violet to sky blue. The results of this study showed that the LAMP method can be conveniently and accurately used to detect the OsGRF4^NM73 gene in rice.

Background/Objectives: With the acceleration of global industrialization and continuous population growth, the world is increasingly confronted with the dual challenges of food insecurity and cultivated land contamination. The screening and breeding of rice varieties with superior agronomic traits and low heavy metal accumulation have therefore become important strategies for ensuring food safety and sustainable agricultural production. Methods: In this study, rice varieties carrying the Gn1a-i gene and exhibiting specific cadmium (Cd) accumulation characteristics were screened using a combination of molecular marker detection and cadmium accumulation evaluation. Specific loop-mediated isothermal amplification (LAMP) primers targeting the Gn1a-i gene were designed and combined with a lateral flow dipstick (LFD) assay to enable rapid genetic screening of rice varieties. A six-day hydroponic experiment under cadmium stress was conducted across three temperature ranges (15–20 °C, 22–27 °C, and 30–35 °C), and cadmium accumulation in different plant organs (roots, stem sheath, and leaves) was analyzed. Results: Seven varieties carrying the Gn1a-i gene, including Xiangwanxian 12, were identified among ten tested rice varieties. Xiangwanxian 12 was subsequently selected for further evaluation, with the high-cadmium-accumulating variety Yuzhenxiang used as a control. At 144 h, the total Cd content in the measured organs of Xiangwanxian 12 was 9.6%, 4.0%, and 23.2% lower than that of Yuzhenxiang under low, medium, and high temperatures, respectively (one-tailed t-test, p < 0.01 for all three temperatures). Conclusions: The integration of LAMP-based genotyping and physiological evaluation provides a novel and reliable strategy for identifying low-Cd rice germplasm. Xiangwanxian 12, which carries the Gn1a-i allele and exhibits consistently lower Cd accumulation than Yuzhenxiang, suggests potential as a candidate for breeding high-yield, low-Cd rice cultivars.