Combined QTL-sequencing, linkage mapping, and RNA-sequencing identify candidate genes and KASP markers for low-temperature germination in Oryza sativa L. ssp. Japonica.

Through QTL-seq, QTL mapping and RNA-seq, six candidate genes of qLTG9 can be used as targets for cold tolerance functional characterization, and six KASP markers can be used for marker-assisted breeding to improve the germination ability of japonica rice at low temperature. The development of direct-seeded rice at high latitudes and altitudes depends on the seed germination ability of rice under a low-temperature environment. However, the lack of regulatory genes for low-temperature germination has severely limited the application of genetics in improving the breeds. Here, we used cultivars DN430 and DF104 with significantly different low-temperature germination (LTG) and 460 F progeny derived from them to identify LTG regulators by combining QTL-sequencing, linkage mapping, and RNA-sequencing. The QTL-sequencing mapped qLTG9 within a physical interval of 3.4 Mb. In addition, we used 10 Kompetitive allele-specific PCR (KASP) markers provided by the two parents, and qLTG9 was optimized from 3.4 Mb to a physical interval of 397.9 kb and accounted for 20.4% of the phenotypic variation. RNA-sequencing identified qLTG9 as eight candidate genes with significantly different expression within the 397.9 kb interval, six of which possessed SNPs on the promoter and coding regions. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) completely validated the results of these six genes in RNA-sequencing. Subsequently, six non-synonymous SNPs were designed using variants in the coding region of these six candidates. Genotypic analysis of these SNPs in 60 individuals with extreme phenotypes indicated these SNPs determined the differences in cold tolerance between parents. The six candidate genes of qLTG9 and the six KASP markers could be used together for marker-assisted breeding to improve LTG.