Grain size is a key factor influencing grain yield and appearance quality in rice. We identified twelve quantitative trait loci (QTL) for grain length (GL), nine for grain width (GW), and nine for 1000-kernel weight (TKW) using GLU-SSSLs, which are single-segment substitution lines with Oryza glumaepatula as donor parent and Huajingxian 74 (HJX74) as recipient parent. Among the QTL, qGL1-2, qGL1-4, qGL9-2, qGW2-2, qGW9-1 and qTKW9-2 contributed to high grain yield. GL9 was identified as a candidate gene for qGL9-2 by map-based cloning and sequencing, and is a novel allele of GS9. The kernel of NIL-gl9 was slenderer and longer than that of HJX74, and the TKW and grain yield per plant of NIL-gl9 were higher than those of HJX74. The proportion of grain chalkiness of NIL-gl9 was much lower than that of HJX74. Thus, gl9 increased grain yield and appearance quality simultaneously. Three pyramid lines, NIL-gs3/gl9, NIL-GW7/gl9 and NIL-gw8/gl9, were developed and the kernel of each was longer than that of the corresponding recipient parent lines. The gl9 allele may be beneficial for breeding rice varieties with high grain yield and good appearance quality.

Progress in plant breeding depends on the development of genetic resources, genetic knowledge, and breeding techniques. The core of plant breeding is the use of naturally occurring variation. At the beginning of the post-genomic era, a new concept of “breeding by design” was proposed, which aims to control all allelic variation for all genes of agronomic importance. In the past two decades, we have applied a three-step strategy for research on rice breeding by design. In the first step, we constructed a single-segment substitution line (SSSL) library using Huajingxian 74 (HJX74), an elite xian (indica) rice cultivar, as the recipient in which to assemble genes from the rice AA genome. In the second step, we identified a series of desirable genes in the SSSL library. In the third step, we designed new rice lines, and achieved the breeding goals by pyramiding target genes in the HJX74-SSSL library. This review introduces the background, concept, and strategy of breeding by design, as well as our achievements in rice breeding by design using the HJX74-SSSL platform. Our practice shows that target chromosome-segment substitution is a way to breeding by design.