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Great yield-enhancing prospects of autotetraploid rice was restricted by various polyploidy-induced reproductive dysfunction. To surmount these challenges, our group has generated a series of valuable fertile tetraploid lines (denoted as neo-tetraploid rice) through 20-year efforts. With this context, a G-type lectin receptor-like kinase, OsNRFG6, was identified as a pivotal factor associated with reproductive regulation in neo-tetraploid rice. Nevertheless, it is still elusive about a comprehensive understanding of its precise functional roles and underlying molecular mechanisms during reproduction of neo-tetraploid rice. Here, we demonstrated that OsNRFG6 executed a constitutive expression pattern and encoded proteins localizing in perinucleus and endoplasmic reticulum. Subsequently, four independent mutant lines of OsNRFG6 within neo-tetraploid rice background were further identified, all displaying low seed-setting rate due to abortive embryo sacs and defective double fertilization. RNA-seq and RT-qPCR revealed a significant down-regulation of OsNRFG6 and female reproductive genes such as OsMEL1 and LOG in ovaries prior to and post-fertilization, attributing this effect to OsNRFG6 mutation. Furthermore, through yeast-two hybrids, bimolecular fluorescence complementation assays, and luciferase complementation imaging assays, it was determined that OsNRFG6 could interact with itself and two female reproductive proteins (LOG and OsDES1) to form protein complexes. These results elucidate the reproductive functions and molecular pathway governed by OsNRFG6 in regulating fertility of neo-tetraploid rice, offering insights into molecular understanding of fertility improvement in polyploid rice. [ABSTRACT FROM AUTHOR]

Background: Neo-tetraploid rice (NTR) is a useful new germplasm that developed from the descendants of the autotetraploid rice (ATR) hybrids. NTR showed improved fertility and yield potential, and produced high yield heterosis when crossed with indica ATR for commercial utilization. However, their classification, population structure and genomic feature remain elusive. Results: Here, high-depth genome resequencing data of 15 NTRs and 18 ATRs, together with 38 publicly available data of diploid rice accessions, were analyzed to conduct classification, population structure and haplotype analyses. Five subpopulations were detected and NTRs were clustered into one independent group that was adjacent to japonica subspecies, which maybe the reason for high heterosis when NTRs crossed with indica ATRs. Haplotype patterns of 717 key genes that associated with yield and other agronomic traits were revealed in these NTRs. Moreover, a novel specific SNP variation was detected in the first exon of HSP101, a known heat-inducible gene, which was conserved in all NTRs but absent in ATRs, 3KRG and RiceVarMap2 databases. The novel allele was named as HSP101–1, which was confirmed to be a heat response factor by qRT-PCR, and knockout of HSP101–1 significantly decreased the thermotolerance capacity of NTR. Interestingly, HSP101–1 was also specifically expressed in the anthers of NTR at pre-meiotic and meiosis stages under optimal environment without heat stress, and its loss-of-function mutant showed significant decrease in fertility of NTR. Conclusion: The construction of first genomic variation repository and the revelation of population structure provide invaluable information for optimizing the designs of tetraploid rice breeding. The detection of specific genomic variations offered useful genomic markers and new directions to resolve high fertility mechanism of NTR. [ABSTRACT FROM AUTHOR]

Background: Neo-tetraploid rice (NTR) is a new tetraploid rice germplasm that developed from the crossing and directional selection of different autotetraploid rice lines, which showed high fertility and promising yield potential. However, systematic yield assessment, genome composition and functional variations associated with fertility and yield remain elusive. Results: Two season's field trials of 15 NTRs and 27 autotetraploid rice (ATR) lines revealed that the improvement of YPP (yield per plant, 4.45 g increase) were significantly associated with the increase of SS (seed setting, 29.44% increase), and yield and seed setting of NTRs improved significantly compared to parental lines. Whole genome resequencing of 13 NTR sister lines and their parents at about 48.63 depth were conducted and genome compositions were illustrated using inherited chromosomal blocks. Interestingly, 222 non-parental genes were detected between NTRs and their low fertility parental lines, which were conserved in 13 NTRs. These genes were overlapped with yield and fertility QTLs, and RNA-Seq analysis revealed that 81 of them were enriched in reproductive tissues. CRISPR/Cas9 gene knockout was conducted for 9 non-parental genes to validate their function. Knockout mutants showed on an average 25.63% and 4.88 g decrease in SS and YPP, respectively. Notably, some mutants showed interesting phenotypes, e.g., kin7l (kinesin motor gene) and kin14m (kinesin motor gene), bzr3 (BES1/BZR1 homolog) and nrfg4 (neo-tetraploid rice fertility related gene) exhibited 44.65%, 24.30%, 24.42% and 28.33% decrease in SS and 8.81 g, 4.71 g, 5.90 g, 6.22 g reduction in YPP, respectively. Conclusion: Comparative genomics provides insights into genome composition of neo-tetraploid rice and the genes associated with fertility and yield will play important role to reveal molecular mechanisms for the improvement of tetraploid rice. [ABSTRACT FROM AUTHOR]