Recently, the Shenzhen Institute of Agricultural Genomics of the Chinese Academy of Agricultural Sciences (Shenzhen Branch of the Guangdong Laboratory of Lingnan Modern Agricultural Science and Technology), together with Yazhou Bay National Laboratory, China Rice Research Institute and other units, published a paper entitled "Population-level exploration of alternative splicing and its unique role in" in the top academic journal of botany, The Plant Cell Controlling agronomic traits of rice", based on the core germplasm resources of wild rice and cultivated rice around the world, this study constructed a population-level, multi-tissue alternative splicing variation map of rice, and mined a number of excellent genes associated with alternative splicing and important agronomic traits, which provides valuable resources and new insights for in-depth understanding of the genetic basis of splice variation and its important regulatory role in controlling rice agronomic traits.
Rice (Oryza sativa) is one of the important food crops, and fully exploring and utilizing the excellent variation resources of rice is an important way to carry out genetic improvement of rice. In the early stage, the team constructed a graph super pan-genome with the largest population size, fully annotated genome and systematic system in rice (Shang et al., 2022), and systematically analyzed a variety of complex structural variants such as transposon variation (Li et al., 2024), inverted variation (He et al., 2024), and centromere sequence variation (Lv et al., 2024) in the rice core germplasm population, and efficiently mined a number of excellent new genes related to high yield and stress resistance in rice. It lays an important foundation for the application of population genetics of rice structural variation. At the same time, combined with the transcriptional expression profile of the super pan-genome population, the key salt-tolerant novel gene STG5 was successfully cloned (Wei et al., 2024). However, there are still a considerable number of phenotypic variations that cannot be directly explained by genome sequence variations, so it is necessary to further explore the potential functional variants in the downstream, and alternative splicing (AS) of functional genes is an important goal.
Alternative splicing is an important post-transcriptional regulatory mechanism in eukaryotes and an important cause of protein diversity, which plays a key role in regulating plant phenotypic variation. However, there are limited studies on AS at the multi-tissue and population levels in rice, and the regulatory effect of AS on agronomic traits is rarely reported, which hinders the potential value of AS in precision breeding and improvement of rice. In this study, the AS profiles of leaf and panicle tissues were constructed using the core germplasm resources of rice (including wild rice and cultivated rice) around the world. Compared with the reference genome, the proportion of AS genes increased by 20%, indicating that the construction of AS maps at the population level increased the diversity of transcripts and provided rich genetic resources for the breeding and improvement of agronomic traits in rice. The results of inter-tissue AS gene comparison showed that 43.7% of AS genes were specifically spliced in leaves or panicles (Fig. 1), indicating that multi-tissue exploration of AS has more comprehensive advantages and necessity. At the same time, this study identified different acclimation events and differential transcripts during indica-japonica differentiation, which are helpful for understanding the changes in gene expression regulation during domestication and differentiation in rice.
Fig.1 Transcript assembly and identification of leaf and panicle tissues in rice populations
In order to elucidate the genetic regulation characteristics of AS, this study used the rice hyper-pangenome (Shang et al., 2022) to link genetic variation with splice phenotypes by splicing quantitative trait locus mapping (sQTL). To explore the effects of single nucleotide polymorphisms (SNPs) and large structural variations (SVs) on gene splicing. The results showed that the effect of cis-regulation was greater than that of trans-regulation, and about 21% of cis-regulation could only be explained by SVs, indicating that large structural variation had an important impact on gene splicing. The tissue-specific genetic regulation of AS was further analyzed, and it was found that only 22.5% of the genes could be detected by sQTL in both leaf and spike tissues, indicating that AS plays an important role in tissue development. In addition, genetic variations that affect gene splicing do not necessarily affect the abundance of overall mRNA, and more than one-third of sGenes (genes with significant sQTLs) do not have significant eQTL signals (Figure 2), suggesting that AS and gene expression may be subject to relatively independent genetic regulation. These findings provide an important reference for in-depth understanding of the genetic regulation of alternative splicing, and also provide an important theoretical basis for future rice breeding and genetic improvement.
Fig. 2 Genetic regulation patterns of sQTLs and eQTLs
In order to explore how AS regulates agronomic traits in rice and its potential value for genetic improvement in rice, splicing-phenotypic association analysis (spQTL) was performed using alternative splicing of genes and ten agronomic traits, and the results showed that 58 splice genes in leaves and 34 splicing genes in panicles were significantly associated with at least one agronomic trait (Fig. 3). Among them, it was found that one plant type trait had the strongest correlation signal with the splicing ratio of OsPIE1 gene, and the correlation with the expression level was not significant. CRISPR/Cas9-mediated OsPIE1 knockout lines were used to significantly change plant type, and a new method of mining agronomic superior genes using AS variants was verified. In conclusion, this study took alternative splicing as the starting point to explore the important regulatory role of alternative splicing on agronomic traits, which provides an important resource for future rice breeding and improvement.
Fig.3 OsPIE1 gene regulates rice plant shape through alternative splicing
Researcher Shang Lianguang from the Shenzhen Institute of Agricultural Genomics of the Chinese Academy of Agricultural Sciences, Qian Qian, academician of Yazhou Bay National Laboratory, and researcher Hu Guanjing from the Institute of Genomics are the co-corresponding authors of the paper. Postdoctoral fellows Hong Zhang, Wu Chen, and De Zhu from the Institute of Genomics, and Bintao Zhang and Qiang Xu, who are currently studying at the master's degree, are the co-first authors of the paper. The research was supported by the Science and Technology Innovation 2030 - Major Project, the Basic Science Center of the National Natural Science Foundation of China, the Outstanding Youth Fund of the Natural Science Foundation of Guangdong Province, the Science and Technology Innovation Engineering Science Center of the Chinese Academy of Agricultural Sciences, and the Youth Innovation Special Fund of the Chinese Academy of Agricultural Sciences. This work was supported by the Institute of Genomics, the China Rice Institute, and the Yazhou Bay Science and Technology City Supercomputing Platform.
Original link: https://doi.org/10.1093/plcell/koae181
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