Yanfeng 47 (YF47) is an elite japonica rice variety cultivated in China on nearly 2 million hectares over the past 20 years. However, YF47 is highly susceptible to rice blast (Magnaporthe oryzae), one of the most destructive rice diseases. In this study, we developed novel TPAP (tetra-primer ARMS-PCR) functional markers for the genes Pita, Pib and Pid2, all of which afford broad-spectrum resistance to blast. A collection of 91 japonica rice germplasms with similar ecological characteristics to YF47 was screened, and Wuyunjing 27 (WYJ27) with Pita and Pib alleles, and P135 with the Pid2 allele, were identified. Furthermore, the corresponding positive Pita, Pib and Pid2 alleles were transferred into YF47 using single, mutual and backcrosses, together with molecular marker-assisted selection (MAS) and anther culture technology. These genetic materials, carrying one, two, or three functional alleles, were generated within three years, and compared to YF47 they all showed improved resistance to naturally inoculated rice blast. Further improved lines (IL) 1 to 5 (all containing Pita, Pib and Pid2 alleles) were evaluated for yield performance, and when no fungicide was applied all lines except IL-4 showed increased traits compared with those of YF47. IL-5, renamed Yanjing 144 (YJ144), showed yield increases in the Liaoning province regional variety comparison test and superior appearance quality compared to YF47. Our work provides a molecular design strategy for pyramiding multiple beneficial genes to rapidly improve rice blast resistance, yield, and quality using multiple breeding strategies.

Background Long grain geng/japonica rice has a higher market preference due its excellent appearance quality. The dense and erect-panicle 1 (dep1) gene has been widely used in the breeding of high-yielding geng/japonica rice cultivars in China. However, this gene causes short and round grain shape thus making it less attractive in global rice markets. Therefore, breeding of high-yielding long-grain geng/japonica rice cultivars by incorporating dep1 with major-effect grain shape gene is of high priority in rice industry. Up to now, multiple grain shape genes’ effect mechanism has been clearly elaborated, however, under the background of erect-panicle geng/japonica rice, the effect of major grain shape genes on the appearance quality need to be further clarified, as detailed reports are limited. Findings: Utilizing CRISPR/Cas9 technology, a series of near-isogenic lines (NILs) (YF47dep1-gw8, YF47dep1-gs3, YF47dep1-gl7, YF47dep1-qgl3 and YF47dep1-tgw6) in Yanfeng 47(YF47dep1) background were created. Grain appearance and yield components analysis showed that: i) All NILs’ grain length to width ratio was significantly increased compared to that of YF47dep1, excepted YF47dep1-gs3, ii) The chalkiness degree was significantly reduced in all of the NILs, iii) In all of the NILs, YF47dep1-gw8 grains exhibited the greatest length to width ratio and the lowest chalkiness degree, iv) The composition of glume cells and filling characteristics of the endosperm were two key factors contributing grain shape and grain chalk variations, respectively, and v) Owning to a substantial increase in the thousand grain weight, the yields of YF47dep1-gs3 and YF47dep1-tgw6 were significantly higher than that of YF47dep1, whereas YF47dep1-qgl3 exhibited the lowest yield because of a dramatic decrease in the effective panicle number and thousand grain weight. Conclusions All the results revealed that pyramiding dep1 with major-effect grain shape alleles was an effective approach to improving the appearance quality of erect-panicle geng/japonica rice, owning to both of the appearance quality and yield improvement, GS3 and TGW6 alleles can be applied directly for breeding long-grain shape geng/japonica rice, and editing GW8 resulted in excellent appearance quality but low yield, therefore, this gene would be difficult to use directly but can be considered as the core germplasm resource.