In this study, one major QTL affecting gel consistency (GC) of japonica/indica cross was identified on chromosome 6 using a DH population. To understand the molecular mechanism that regulates GC in rice grains, the major QTL (qGC-6) was isolated through a map-based cloning approach utilizing chromosome segment substitution lines (CSSLs). Using 64 plants with extremely soft GC that were selected on recombinant break points between two SSR markers, RM540 and RM8200 in a BC4F2 population, qGC-6 was mapped to a 60-kb DNA region between two STS markers, S26 and S27. These two markers were then used to further identify recombination break points. Finally, qGC-6 was delimited in an interval of a 11-kb region. Gene prediction analysis of the 11-kb DNA sequence containing qGC-6 identified only one putative ORF, which encodes granule-bound starch synthesis protein (Wx protein). Results of sequencing analysis and complementation experiment confirmed that this candidate ORF is responsible for rice GC. Genetic evidences revealed that Wx might contribute equally to the grain amylose content-controlling gene as well as gel consistency. This new information is important to breed rice varieties with improved grain quality.

The percentage of amylose in the endosperm of rice (Oryza sativa) largely determines grain cooking and eating qualities. Granule‐bound starch synthase I (GBSSI) and GBSSII are responsible for amylose biosynthesis in the endosperm and leaf, respectively. Here, we identified OsGBP, a rice GBSS‐binding protein that interacted with GBSSI and GBSSII in vitro and in vivo. The total starch and amylose contents in osgbp mutants were significantly lower than those of wild type in leaves and grains, resulting in reduced grain weight and quality. The carbohydrate‐binding module 48 (CBM48) domain present in the C‐terminus of OsGBP is crucial for OsGBP binding to starch. In the osgbp mutant, the extent of GBSSI and GBSSII binding to starch in the leaf and endosperm was significantly lower than wild type. Our data suggest that OsGBP plays an important role in leaf and endosperm starch biosynthesis by mediating the binding of GBSS proteins to developing starch granules. This elucidation of the function of OsGBP enhances our understanding of the molecular basis of starch biosynthesis in rice and contributes information that can be potentially used for the genetic improvement of yield and grain quality.

Under conditions of labor or resource scarcity, direct seeding, rather than transplantation, is the preferred mode of rice (Oryza sativa) cultivation. This approach requires varieties that exhibit uniform seedling emergence. Mesocotyl elongation (ME), the main driver of rapid emergence of rice seedlings from soil, is enhanced by darkness and inhibited by light. Plant polyamine oxidases (PAOs) oxidize polyamines (PAs) and release H 2 O 2 . Here, we established that OsPAO5 expression in rice seedlings is increased in the presence of light and inhibited by darkness. To determine its role in ME, we created OsPAO5 mutants using CRISPR/Cas9. Compared with the wild type, pao5 mutants had longer mesocotyls, released less H 2 O 2 , and synthesized more ethylene. The mutant seedlings emerged at a higher and more uniform rate, indicating their potential for use in direct seeding. Nucleotide polymorphism analysis revealed that an SNP (PAO5 À578G/A ) located 578 bp upstream of the OsPAO5 start codon alters its expression, and was selected during rice mesocotyl domestication. The PAO5 À578G genotype conferring a long mesocotyl mainly exists in wild rice, most Aus accessions, and some Geng (Japonica) accessions. Intriguingly, knocking out OsPAO5 can remarkably increase the grain weight, grain number, and yield potential. In summary, we developed a novel strategy to obtain elite rice with higher emergence vigor and yield potential, which can be conveniently and widely used to breed varieties of direct-seeding rice.

No abstract

Aroma is a key grain quality trait that directly influences the market price of rice globally. Loss of function of betaine aldehyde dehydrogenase 2 (OsBADH2) affects the biosynthesis of 2-acetyl-1pyrroline (2-AP), which is responsible for aroma in fragrant rice. The current study was aimed at creating new alleles of BADH2 using CRISPR/Cas9 gene editing technology under the genetic background of the japonica Ningjing 1 (NJ1) and indica Huang Huazhan (HHZ) varieties. Sensory evaluation and analysis using headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) showed that the grains of the four homozygous T 1 lines with new alleles of BADH2 (nj1-cr BADH2 -1, nj1-cr BADH2 -2, hhz-cr BADH2 -1 and hhz-cr BADH2 -2) produced moderate fragrance and had significantly increased 2-AP content compared with wild-types. Moreover, there were no significant differences in the amylose content and gelatinization temperature among the four lines with new alleles of BADH2 to the wild-types. Thereafter, we crossed the HHZ background new alleles of BADH2 with CMS line Taonong 1A (TN1A) to produce a three-line hybrid variety B-Tao-You-Xiangzhan (BTYXZ) with increased grain aroma. The 2-AP content in grains of the improved BTYXZ-1 and BTYXZ-2 reached at 26.16 and 18.74 μg/kg, and the gel consistency of BTYXZ-1 and BTYXZ-2 increased significantly by 9.1% and 6.5%, respectively, compared with the wild-type Tao-You-Xiangzhan (TYXZ). However, the γaminobutyric acid (GABA) content in the improved three-line hybrid rice BTYXZ-1 (5.6 mg/100 g) and BTYXZ-2 (10.7 mg/100 g) was significantly lower than that of the TYXZ. These results demonstrated that CRISPR/Cas9 gene editing technology could be successfully utilized in improving aroma in non-fragrant japonica and indica varieties. In addition, the newly developed BADH2 alleles provided important genetic resources for grain aroma improvement in three-line hybrid rice.