关键词: 水稻, 抽穗期, DTH8/Ghd8, BSA-seq, 进化

Abstract:

【Objective】 The heading date plays a crucial role in influencing the regional adaptation and yield of rice (Oryza sativa L.). The identification of early heading genes contributes significantly to enhancing and fine-tuning the regulatory network that controls rice heading, which provides valuable genetic resources for molecular breeding with the goal of achieving early maturity and high yield in rice. 【Method】 CL33, a chromosome segment substitution line with early heading, and 93-11, its recipient parent with late heading, were used as research materials to investigate and analyze their major agronomic traits. Two DNA pools were constructed, comprising plants exhibiting extremely early and late heading. Whole-genome resequencing and BSA-Seq analyses were then conducted to locate the genomic region associated with the heading date. In the subsequent steps, InDel markers within this identified region were developed for fine mapping. The gene LOC-Os08g07740 emerged as the primary candidate gene within localization intervals, determined through gene prediction, candidate gene analyses, as well as references to relevant literatures. This candidate gene was subsequently cloned and analyzed for allelic variations. Moreover, we explored the genetic and phylogenetic relationships of the LOC_Os08g07740 gene within the three rice subgroups, Indica, Japonica and O. rufipogon. This analysis involved studying genomic data within approximately 40 kb upstream and downstream of the gene utilizing bioinformatics software.【Result】 Under both natural long-day and short-day conditions in Nanning, Guangxi, CL33 exhibited a 20-25 days shorter than its recipient parent 93-11. Moreover, under natural long-day conditions, the agronomic traits of CL33 were largely similar to those of 93-11, with the exception of a shortened spike length and a reduced number of grains per spike. Genetic analysis revealed that the early heading trait in CL33 was controlled by a recessive gene. This gene was finely localized within a 100 kb region between the markers PSM8-6 and PSM8-8 on the short arm of rice chromosome 8, encompassing 15 predicted candidate genes. Significantly, the candidate gene ORF13 (LOC_Os08g07740), which shared alleles with known heading date genes like DTH8/Ghd8 emerged as a key candidate. Sequencing and sequence alignment of ORF13 demonstrated an 888 bp coding sequence without introns, encoding a protein of 295 amino acids. Compared to the recipient parent 93-11, LOC_Os08g07740 in CL33 featured a 6 bp insertion and a 9 bp deletion between the 535-536th and 820-821st base pairs, respectively, resulting in consequential amino acid sequence alterations. Hence, it was tentatively named OsEHD8 as the target candidate gene. Genetic evolutionary analyses indicated a significant decrease in genetic diversity within the LOC_Os08g07740 gene in Indica and Japonica compared to O. rufipogon, with a 62.53% decrease in Indica and a 53.76% decrease in Japonica. Nevertheless, the differences in genetic diversity between Indica and Japonica were not statistically significant. The LOC_Os08g07740 gene featured a total of 13 haplotypes, with the Hap_2 possibly representing the common ancestor of the three subgroups. Geographical isolation or environmental differences might have led to the fixation of different haplotypes in the Indica and Japonica subgroups. These findings suggested that the LOC_Os08g07740 gene underwent directional selection in the three subgroups.【Conclusion】 OsEHD8, identified as a functional allele of DTH8/Ghd8, played a key role in promoting early heading in rice under both natural long-day and short-day conditions. Moreover, the chromosomal segment substitution line CL33, which carried the OsEHD8 allele, exhibited no significant differences in other agronomic traits compared to the recipient parent 93-11 under natural long-day conditions, except for a shorter spike length and a reduction in grains per spike.

Key words: rice (Oryza sativa L.), heading date, DTH8/Ghd8, BSA-seq, evolution