Targeted manipulation of grain shape genes effectively improves outcrossing rate and hybrid seed production in rice

Stigma exsertion rate (SER) of the male sterile line is a key limiting factor for hybrid seed production in rice. Although a large number of quantitative trait loci associated with SER have been reported, few genes have been molecularly cloned and functionally characterized, severely hindering the genetic improvement of SER of the male sterile line and the breeding efficiency of hybrid rice. In this study, we identified three grain shape regulatory genes, GS3, GW8 and GS9, as potential candidate genes for targeted manipulation of grain shape and SER. We show that simultaneously knocking out these three genes could effectively increase SER by increasing the ratio of spikelet length/spikelet width and length of stigma and style, without negative impacts on other agronomic traits. Cellular examination and transcriptomic analyses revealed a role of these genes in coordinated regulation of transverse and longitudinal cell division in the pistils. Moreover, we demonstrate that targeted manipulation of these grain shape genes could significantly improve the outcrossing rate in both the ZH11 (a japonica variety) and Zhu6S (an indica male sterile line) backgrounds. Our results provide new insights into the mechanisms of rice SER regulation and develop an effective strategy to improve SER and out-crossing rate in rice, thus facilitating hybrid rice production.