Identifying a large number of high-yield genes in rice by pedigree analysis, whole-genome sequencing, and CRISPR-Cas9 gene knockout

Abstract

Repeated artificial selection of a complex trait facilitates the identification of genes underlying the trait, especially if multiple selected descendant lines are available. Here we developed a pedigree-based approach to identify genes underlying the Green Revolution (GR) phenotype. From a pedigree analysis, we selected 30 cultivars including the "miracle rice" IR8, a GR landmark, its ancestors and descendants, and also other related cultivars for identifying high-yield genes. Through sequencing of these genomes, we identified 28 ancestral chromosomal blocks that were maintained in all the high-yield cultivars under study. In these blocks, we identified six genes of known function, including the GR gene sd1, and 123 loci with genes of unknown function. We randomly selected 57 genes from the 123 loci for knockout or knockdown studies and found that a high proportion of these genes are essential or have phenotypic effects related to rice production. Notably, knockout lines have significant changes in plant height (P < 0.003), a key GR trait, compared with wild-type lines. Some gene knockouts or knockdowns were especially interesting. For example, knockout of Os10g0555100, a putative glucosyltransferase gene, showed both reduced growth and altered panicle architecture. In addition, we found that in some retained chromosome blocks several GR-related genes were clustered, although they have unrelated sequences, suggesting clustering of genes with similar functions. In conclusion, we have identified many high-yield genes in rice. Our method provides a powerful means to identify genes associated with a specific trait.

Keywords: Green Revolution; gene knockout; high-yield gene; pedigree analysis.

Fig. 1.

Pedigree and flowchart for the identification of gene loci under selection. (A) An abridged pedigree of the major rice cultivars used in this study. The cultivars shown in blue boxes were resequenced; cultivars shown in gray boxes were not. “OP” means “the other parent”; cultivars so identified were not sequenced. Percentages in boxes show the expected probability of a given locus being inherited from DGWG (D) or Peta (P) in that generation. The bottom box indicates the expected probabilities that a locus is shared by all eight MH63 descendants, which are extremely low (SI Appendix, Table S4). A solid arrow denotes a direct parent (i.e., IR24), and a dashed arrow indicates an indirect ancestor (i.e., IR20). (B) Flowchart of the approach used to identify candidate blocks and gene loci derived from DGWG or Peta. Numbers of blocks (B) and gene loci (G) within the high-confidence blocks are shown in each step of filtering. The six reported genes (three from DGWG and three from Peta) are the gene loci that have clear functions reported in literature. Most of the 129 gene loci contain only one gene, but 28 loci contain two or more overlapped genes (Methods).

Fig. 2.

Blocks inherited from DGWG and Peta in IR8, IR24, IR30, MH63, and the eight descendants of MH63. Blue and red bars represent blocks derived from DGWG and Peta, respectively. “Shared” denotes the regions shared in all eight MH63 descendants. The purple arrows represent the six genes reported with functions related to plant type or high yield; asterisks represent the 123 gene loci with unknown functions; the six genes are shared by all eight MH63 descendants and five collateral series. Chromosomes 3, 4, and 6, which contain no regions shared by all eight MH63 descendants, are shown in SI Appendix, Fig. S7. The next-to-last block on chromosome 1 was shortened using breaks.

Fig. 3.

Photographs of knockout mutants with changed phenotypes compared with WT Kasalath. These six examples show shorter plants (A and B), rolling leaves (C), a later heading date (D), changed panicles (E), and empty seeds (F) in the mutants compared with WT plants. The other nine knockout mutants with observable phenotypic changes and the controls are shown in SI Appendix, Fig. S8.