Identification of QTL for kernel number-related traits in a rice chromosome segment substitution line and fine mapping of qSP1

A chromosome segment substitution line (CSSL) is a powerful tool for combining quantitative trait locus (QTL) mapping with the pyramiding of desirable alleles. The rice CSSL Z1364 with increased kernel number was identified in a BC3F8 population derived from a cross of Nipponbare as the recipient with Xihui 18 as the donor parent. Z1364 carried three substitution segments distributed on chromosomes 1, 6, and 8. The mean substitution length was 1.19 Mb. Of 17 QTL identified on the substitution segments, qSP1 for spikelets per panicle, qSSD1 for seed-set density, and qNSB1 for number of secondary branches explained respectively 57.34%, 87.7%, and 49.44% of the corresponding phenotypic variance and were all linked to RM6777. Chi-square analysis showed that the increased kernel number in Z1364 was inherited recessively by a single gene. By fine mapping, qSP1 was delimited to a 50-kb region on the short arm of chromosome 1. Based on DNA sequence, a previously uncharacterized rice homolog of Arabidopsis thaliana AT4G32551 was identified as a candidate gene for qSP1 in which mutation increases the number of spikelets and kernels in Z1364. qSP1 was expressed in all tissues, but particularly in 1-cm panicles. The expression levels of OsMADS22, GN1A, and DST were upregulated and those of LAX2, GNP1, and GHD7 were downregulated in Nipponbare. These results provide a foundation for functional research on qSP1.

Auxin signaling module OsSK41-OsIAA10-OsARF regulates grain yield traits in rice

Auxin is an important phytohormone in plants, and auxin signaling pathways in rice play key roles in regulating its growth, development, and productivity. To investigate how rice grain yield traits are regulated by auxin signaling pathways and to facilitate their application in rice improvement, we validated the functional relationships among regulatory genes such as OsIAA10, OsSK41, and OsARF21 that are involved in one of the auxin(OsIAA10) signaling pathways. We assessed the phenotypic effects of these genes on several grain yield traits across two environments using knockout and/or overexpression transgenic lines.Based on the results, we constructed a model that showed how grain yield traits were regulated by OsIAA10 and OsTIR1, OsAFB2, and OsSK41 and OsmiR393 in the OsSK41-OsIAA10-OsARF module and by OsARF21 in the transcriptional regulation of downstream auxin response genes in the OsSK41-OsIAA10-OsARF module. The population genomic analyses revealed rich genetic diversity and the presence of major functional alleles at most of these loci in rice populations. The strong differentiation of many major alleles between Xian/indica and Geng/japonica subspecies and/or among modern varieties and landraces suggested that they contributed to improved productivity during evolution and breeding. We identified several important aspects associated with the genetic and molecular bases of rice grain and yield traits that were regulated by auxin signaling pathways.We also suggested rice auxin response factor(OsARF) activators as candidate target genes for improving specific target traits by overexpression and/or editing subspecies-specific alleles and by searching and pyramiding the ‘best' gene allelic combinations at multiple regulatory genes in auxin signaling pathways in rice breeding programs.

The kinase OsSK41/OsGSK5 negatively regulates amylose content in rice endosperm by affecting the interaction between OsEBP89 and OsBP5

Amylose content(AC) is the main factor determining the palatability, viscosity, transparency, and digestibility of rice(Oryza sativa)grains. AC in rice grains is mainly controlled by different alleles of the Waxy(Wx) gene. The AP2/EREBP transcription factor OsEBP89 interacts with the MYC-like protein OsBP5 to synergistically regulate the expression of Wx.Here, we determined that the GLYCOGEN SYNTHASE KINASE 5(OsGSK5, also named SHAGGY-like kinase 41 [OsSK41]) inhibits the transcriptional activation activity of OsEBP89 in rice grains during amylose biosynthesis. The loss of OsSK41 function enhanced Wx expression and increased AC in rice grains. By contrast, the loss of function of OsEBP89 reduced Wx expression and decreased AC in rice grains. OsSK41 interacts with OsEBP89 and phosphorylates four of its sites(Thr-28,Thr-30, Ser-238, and Thr-257), which makes OsEBP89 unstable and attenuates its interaction with OsBP5. Wx promoter activity was relatively weak when regulated by the phosphomimicvariantOsEBP89E–OsBP5but relatively strong when regulated by the nonphosphorylatable variant OsEBP89A–OsBP5.Therefore, OsSK41-mediated phosphorylation of OsEBP89 represents an additional layer of complexity in the regulation of amylose biosynthesis during rice grain development. In addition, our findings provide four possible sites for regulating rice grain AC via precise gene editing.