Development and application of two novel functional molecular markers of BADH2 in rice

BACKGROUND: Fragrance is one of the most important quality traits in rice, and the phenotype is attributed to the loss-of-function betaine aldehyde dehydrogenase (BADH2) gene. At least 12 allelic variations of BADH2 have been identified, and some of these have been applied to rice fragrance breeding using traditional molecular markers and Sanger sequencing techniques. However, these traditional methods have several limitations, such as being very expensive, imprecise, inefficient, and having security issues. Thus, a new molecular marker technology must be developed to improve rice fragrance breeding. RESULTS: In this study, more than 95% of the cultivated fragrant rice varieties belonged to a 7-bp deletion in exon 2 (badh2-E2) or an 8-bp deletion and 3-bp variation in exon 7 (badh2-E7). Both allelic variations resulted in the loss of function of the badh2 gene. We developed two novel SNP molecular markers, SNP_badh2-E2 and SNP_badh2- E7, related to the alleles. Their genotype and phenotype were highly cosegregated in the natural variation of rice accessions, with 160 of the 164 fragrant rice varieties detected with the two markers. These markers cosegregated with the fragrance phenotype in the F2 population. CONCLUSIONS: Two functional SNP molecular markers of badh2-E2 and badh2-E7 allelic variations were developed. These functional SNP molecular markers can be used for genotype and genetic improvement of rice fragrance through marker-assisted selection and will significantly improve the efficiency of fragrant rice breeding and promote commercial molecular breeding of rice in the future.

The OsmiR396c-OsGRF4-OsGIF1 regulatory module determines grain size and yield in rice.

Grain weight is the most important component of rice yield and is mainly determined by grain size, which is generally controlled by quantitative trait loci (QTLs). Although numerous QTLs that regulate grain weight have been identified, the genetic network that controls grain size remains unclear. Herein, we report the cloning and functional analysis of a dominant QTL, grain length and width 2 (GLW2), which positively regulates grain weight by simultaneously increasing grain length and width. The GLW2 locus encodes OsGRF4 (growth-regulating factor 4) and is regulated by the microRNA miR396c in vivo. The mutation in OsGRF4 perturbs the OsmiR396 target regulation of OsGRF4, generating a larger grain size and enhanced grain yield. We also demonstrate that OsGIF1 (GRF-interacting factors 1) directly interacts with OsGRF4, and increasing its expression improves grain size. Our results suggest that the miR396c-OsGRF4-OsGIF1 regulatory module plays an important role in grain size determination and holds implications for rice yield improvement.

ROLLED LEAF 9, encoding a GARP protein, regulates the leaf abaxial cell fate in rice.

Leaves, the collective organ produced by the shoot apical meristem (SAM), are polarized along their adaxial-abaxial axis. In this study, we characterized two rice (Oryza sativa) allelic rolled-leaf mutants, rolled leaf 9-1 (rl9-1) and rl9-2, which display very similar phenotypes with completely adaxialized leaves and malformed spikelets. We cloned the RL9 gene by way of a map-based cloning strategy. Molecular studies have revealed that RL9 encodes a GARP protein, an orthologue of Arabidopsis KANADIs. RL9 is mainly expressed in roots, leaves, and flowers. The transient expression of a RL9-GFP (green fluorescent protein) fusion protein has indicated that RL9 protein is localized in the nucleus, suggesting that RL9 acts as a putative transcription factor.

Fine mapping and isolation of Bc7(t), allelic to OsCesA4.

Several brittle culm mutants of rice were identified and characterized. In this study, we characterized a brittle mutant (bc7(t)) identified from japonica variety Zhonghua 11 by means of 60Co-gamma radiation. This mutant displays normal phenotype similar to its wild type plants except for the fragility of all plant body, with approximately 10% decrease in the cellulose content. The genetic analysis and gene fine mapping showed that the bc7(t) mutant was controlled by a recessive gene, residing on an 8.4 kb region of the long arm of chromosome 1. The gene annotation indicated that there was only one putative gene encoding cellulose synthase catalytic subunit (CesA) in this region, which was allelic to OsCesA4. Furthermore, the sequence analysis was carried out and 7 bases deletion in the junction of exon 10 and intron 10 was done in bc7(t) mutant, resulting in the change of reading frame and the consequent failure to generate functional protein. In addition, the result of RNA interference experiment showed that when the Bc7(t) was knocked down, the transplants exhibited fragility, similar to bc7(t) mutant. The finding of novel allele of OsCesA4 locus will facilitate the understanding of the mechanism of cell wall biosynthesis. The potential utilization of the bc7(t) mutant in animal food was discussed as well.