Recently, a team of researchers liu Luxiang from the Institute of Crop Sciences of the Chinese Academy of Agricultural Sciences published a research paper entitled "Cloning and functional characterization of Rht8, a "Green Revolution" replacement gene in wheat" at Molecular Plant. In this study, two dwarf stalk mutants were constructed separately, and the important dwarf stalk gene Rh8 widely used in wheat breeding was successfully cloned, and the mechanism of Rhht8 regulating plant height was initially revealed by using gibberellin spraying, content determination and gene expression.
In the 1960s and 1970s, the use of wheat dwarf genes Rh-B1b and Rh-D1b significantly improved lodging resistance and harvest index, thereby greatly increasing grain production and triggering the famous "Green Revolution". However, Rht-B1b and Rhht-D1b are gibberellin insensitive, resulting in shorter germ sheaths, affecting the germ-germing ability of seed top soil and seedling viability, etc., limiting their further breeding and utilization. The wheat gibberellin-sensitive dwarf stalk gene Rh8 has been widely used in southern and central Europe since the 1930s by Japanese red wheat. Wheat seedling embryo sheath with Rh8 dwarf stalk gene has a long sheath and high growth vitality, which is suitable for growth in high temperature and dry environment, which effectively makes up for the lack of green revolution dwarf stalk gene source. Although there have been international reports on the localization of the Rht8 gene, cloning the Rht8 gene has always been a problem due to the complexity of the wheat genome.
The team screened two excellent dwarf stalk mutants, Rh8-2 and Rht8-3 (Figure 1A-C), from the wheat mutant library and other yield-related indicators such as 1,000 grain weight and panicle number. By hybridizing with the wild type, two large genetic isolation populations were constructed, and the Rh8 gene was located in the physical range of about 700 kb and 170 kb of 2D chromosome, respectively. This segment had a large unknown sequence region in the Chinese Spring Reference Genome at that time, and further assembled wild-type Jing 411 by three-generation sequencing, and found that the gene encoding the RNase H domain formed 2 tandem copies with a functional type and another mutant type, located in this localization segment (Figure 1D). However, the latest Fielder reference genome shows that the gene is 3 tandem copies, so the specific copy number needs to be further experimentally verified. Gene cloning and sequencing analysis found that the gene produced a code shift and an early termination mutation in the two mutants, respectively, ultimately resulting in an incomplete protein encoding (Figure 1E).
Figure 1: Atlas clone of the Rht8 dwarf stalk gene in wheat.
Gene editing knockout of the candidate gene found that the edited plant height was significantly reduced (Figure 2A), and sequencing analysis of the Japanese red wheat cultivar derived from Rh8 found that the mutation site of Rh8-2 was consistent with that of red wheat, and there were differences between red wheat and Rh8-2 mutants in other loci, which showed that the Rh8-2 mutation site was exactly the dwarf stalk site produced by the natural variation of Rh8 gene. Gene expression analysis of different tissues shows that the Rht8 gene is mainly expressed in the nodes of the stem and has a significantly reduced expression in the Rh8-2 mutant (Figure 2B). Subcellular localization analysis of wheat protoplasts found that the wild-type Rh8 protein was localized in the nucleus (Figure 2C), suggesting a transcriptional regulatory role. Gibberellin spraying, content determination and expression analysis showed that Rht8 reduced the content of active gibberellin GA3 and increased the content of GA4 by regulating the expression of genes such as GIBBROMYCIN synthesis related genes GA13ox and GA20ox-2 ,thereby reducing the plant height (Figure 2D-F). Near-isogenic line analysis found that Rht8 significantly reduced plant height without affecting thyme weight and panicle number (Figure 2G-I). At the same time, the study also revealed that Rht8 has a higher distribution frequency in Chinese wheat varieties, especially in the Huanghuai wheat region, and that the Rht8 dwarf stalk genotype evolved after wheat hexagonalization.
Fig. 2: Mechanism analysis and analysis of near-isogen lines of Rht8 regulatory plant height.
The cloning of rht8 gene is of great significance for wheat dwarf breeding and the cultivation of new wheat varieties with suitable plant height. The development of genetic functional molecular markers can realize the accurate use of Rh8 gene breeding, while accelerating the breeding process of wheat plants with high improvement. In addition, this study also provides an important theoretical reference for the study of the mechanism of high regulation of wheat plants.
While the team independently carried out rh8 gene cloning, Professor Ni Zhongfu's team of China Agricultural University also obtained similar conclusions to this study using natural variation materials. The results were published in the journal Molecular Plant in back-to-back form at the same time.
Zhou Chunyun, associate researcher and doctoral student of the Institute of Science and Technology, and Fu Meiyu, a master's student at the Institute of Sciences, are the co-first authors of the paper, and Researcher Liu Luxiang of the Institute of Science and Technology is the corresponding author of the paper. Wang Ke, associate researcher of the Institute of Science and Technology, and Professor Li Xuejun of Northwest A & F University participated in some of the research work. This research has been funded by the National Key R&D Program, the National Natural Science Foundation of China and the Science and Technology Innovation Project of the Chinese Academy of Agricultural Sciences.
Original link:
https://www.cell.com/molecular-plant/fulltext/S1674-2052(22)00014-4
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