Key message
A minor QTL for grain weight in rice, qTGW1.2b, was fine-mapped. Its casual gene OsVQ4 was confirmed through CRISPR/Cas9-targeted mutagenesis, exhibiting an effect that was larger than the original QTL effect.
Abstract
The CRISPR/Cas system exhibits a great potential for rice improvement, but the application was severely hindered due to insufficient target genes, especial the lack of validated genes underlying quantitative trait loci having small effects. In this study, a minor QTL for grain weight, qTGW1.2b, was fine-mapped into a 44.0 kb region using seven sets of near isogenic lines (NILs) developed from the indica rice cross (Zhenshan 97)3/Milyang 46, followed by validation of the causal gene using CRISPR/Cas9-targeted mutagenesis. In the NIL populations, 1000-grain weight of the Zhenshan 97 homozygous lines decreased by 0.9–2.0% compared with the Milyang 46 homozygous lines. A gene encoding VQ-motif protein, OsVQ4, was identified as the candidate gene based on parental sequence differences. The effect of OsVQ4 was confirmed by creating CRISPR/Cas9 knockout lines, whose 1000-grain weight decreased by 2.8–9.8% compared with the wild-type transgenic line and the recipient. These results indicate that applying genome editing system could create novel alleles with large phenotypic variation at minor QTLs, which is an effective way to validate causal genes of minor QTLs. Our study establishes a strategy for cloning minor QTLs, which could also be used to identify a large number of potential target genes for the application of CRISPR/Cas system.
Chlorophyll, one of the major chloroplast components for photosynthesis, has a positive relationship with the photosynthetic rate. The chlorophyll content is an important assessment parameter in agronomy and plant biology research. This study was conducted to evaluate the natural variation in the chlorophyll content and to determine the differential response of the chlorophyll concentration to dark treatment in a natural population containing 139 maize inbreds. A five-fold higher chlorophyll concentration was measured in the light compared with the dark. Meanwhile, the wide variation in the chlorophyll concentration showed the differential response of the natural maize population to dark. Finally, we identified some inbreds that were highly sensitive to the dark with more than 70% difference between the light and dark treatment, such as Dan598, Zheng29, Zheng35, DH29, and R08, as well as some inbreds that had lower sensitivity to the dark, with less than 35% difference in the chlorophyll content between the light and dark treatment, such as Chuan48-2, 4F1, 303WX, 9642, and LY042.
Grain size is the major determinant of grain weight, and a trait having an important role in grain quality. It is controlled by several major quantitative trait loci (QTL) and many minor QTL. Identification of QTL for grain size is important for understanding the genetic and molecular network regulating grain size in rice. Following previous identification of QTL for grain weight and size using an F 2:3 family derived from the indica rice cross Teqing/IRBB52, one QTL, qTGW5 having significant effects on grain length and weight was targeted for validation, dissection and fine-mapping. Firstly, the effect of qTGW5 was validated using two near-isogenic line (NIL)-F 2 populations. Then, qTGW5 was dissected into two closely linked QTL using four NIL populations. One of them, qGL5.1, having significant effects on grain length, width and weight, was located within an 1896.4-kb region. The other one, qGL5.2 controlling grain length, was further delimited into a 68.8-kb region using seven NIL-F 2 populations. Six annotated genes were found in the qGL5.2 region, of which five showed nucleotide polymorphisms between the two parental lines. Additionally, three of the six annotated genes showed significant expression differences between NIL Teqing and NIL IRBB52 in young panicles using qRT-PCR. The results will facilitate cloning the minor QTL and understanding the genetic architecture for grain size in rice.
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