Functional characterization and allelic mining of OsGLR genes for potential uses in rice improvement

Abstract

Glutamate-like receptor (GLR) genes are a group of regulatory genes involved in many physiological processes of plants. With 26 members in the rice genome, the functionalities of most rice GLR genes remain unknown. To facilitate their potential uses in rice improvement, an integrated strategy involving CRISPR-Cas9 mediated knockouts, deep mining and analyses of transcriptomic responses to different abiotic stresses/hormone treatments and gene CDS haplotype (gcHap) diversity in 3,010 rice genomes was taken to understand the functionalities of the 26 rice GLR genes, which led us to two conclusions. First, the expansion of rice GLR genes into a large gene family during evolution had gone through repeated gene duplication events occurred primarily in two large GLR gene clusters on rice chromosomes 9 and 6, which was accompanied with considerable functional differentiation. Secondly, except for two extremely conserved ones (OsGLR6.2 and OsGLR6.3), rich gcHap diversity exists at the remaining GLR genes which played important roles in rice population differentiation and rice improvement, evidenced by their very strong sub-specific and population differentiation, by their differentiated responses to day-length and different abiotic stresses, by the large phenotypic effects of five GLR gene knockout mutants on rice yield traits, by the significant association of major gcHaps at most GLR loci with yield traits, and by the strong genetic bottleneck effects and artificial selection on the gcHap diversity in populations Xian (indica) and Geng (japonica) during modern breeding. Our results suggest the potential values of the natural variation at most rice GLR loci for improving the productivity and tolerances to abiotic stresses. Additional efforts are needed to determine the phenotypic effects of major gcHaps at these GLR loci in order to identify ‘favorable’ alleles at specific GLR loci specific target traits in specific environments to facilitate their application to rice improvement in future.

Functional differentiation and genetic diversity of rice cation exchanger (CAX) genes and their potential use in rice improvement

“…In contrast, OsCAX1b , OsCAX1c , OsCAX3 and OsCAX4 each was expected to be of potential value in rice improvement because of their associations with specific rice traits, their responsiveness to specific abiotic stresses or phytohormones, and relatively high gcHap and CRE diversity. However, additional efforts have to be taken to characterize and verify the ‘desirable’ allele(s) and their phenotypic effects at each of these OsCAX genes, which can be easily achieved by the CRISPR-9 mediated knockout or knock-in mutagenesis experiments 48 .…”

“…To understand how modern breeding in past decades affected the gcHap diversity of rice CAX genes, we compared the differences in E H estimates and frequencies of the predominant gcHaps between 732 Xian ( indica ) landraces (LANs- Xian ) and 328 modern Xian varieties (MVs- Xian ) and between 358 Geng ( japonica ) landraces (LANs- Geng ), and 139 modern Geng varieties (MVs- Geng ) ( https://www.rmbreeding.cn/Index) 47 using an R script ( https://github.com/isaac-Tsang/gcHap_diversity_LANs_MVs -) and Z tests 27 , 48 . We also compared the differences between LANs- Xian and MVs- Xian and between LANs- Geng and MVs- Geng for possible newly emergent gcHaps at each of the CAX genes in rice MV varieties.…”

Identification of the CNGC Gene Family in Rice and Mining of Alleles for Application in Rice Improvement

“…Nei’s genetic identity (INei) was used to measure the genetic similarity between two populations or individuals based on their gcHap frequencies at different OsCNGC loci [ 29 ]. The formulas used to calculate EH and INei referred to the methodology described in a previous study [ 11 ], where OsCNGC genes were classified into conserved genes based on gcHap diversity and subspecies/population differentiation (EH < 0.3 in 3KRG and INei ≥ 0.8 between pairwise populations), and Xian-Geng differentiated genes ( X-G ) (EH < 0.3 in both Xian and Geng and INei ≤ 0.2 between Xian and Geng), and other genes [ 17 ]. To understand how modern breeding over the past decades has affected the gcHap diversity of OsCNGC genes, we collected detailed information on a total of 3010 3KRG rice materials.…”

“…However, the rapid progress in functional and population genomic studies in rice has not yet been widely applied to the development of more efficient breeding techniques. This is due to the fact that there is incomplete information on the phenotypic effect(s) of cloned genes because most experiments were conducted in the laboratory instead of breeding target environments, and therefore, the effects of environmentally interacting genotypes and genetic backgrounds of most cloned genes on some important agronomic traits are largely uncertain [ 17 ]. Moreover, before spending much effort and funds to study a specific gene of interest, researchers should check whether similar alleles have been identified or fixed in commercial crop varieties; and if a gene has been studied for decades, it is unlikely to contribute to a sudden significant increase in yield [ 20 ].…”

“…Rice is one of the most important cereal crops in the world as well as a cereal model plant with high intraspecific genetic diversity [15,16]. In rice, more than 4600 genes have been cloned and functionally characterized at the molecular level (https://funricegenes.git hub.io/ (accessed on 26 September 2023)) [17]. The primary gene pool was found to have extremely high genomic diversity by sequencing 3010 different rice materials (3KRG) from 89 countries worldwide [18,19].…”