Title : Fragrance in rice (Oryza sativa L.) caused by natural mutants and CRISPR/Cas9 mutagenesis
Abstract:
Rice fragrance is one of the most highly valued traits, commanding higher prices on the global market. Basmati and Thai Jasmine rice are the two most popular aromatic rice varieties that are sold at high prices in the world market. The 2-acetyl-1-pyrroline (2AP) is regarded as the major aromatic compound that contributes to the fragrance in many of the aromatic rice varieties, although 100s of other volatile compounds are reported at minor levels. The causal gene for 2AP type fragrance has been well documented as a mutation occurred in Badh2 gene that causes a nonfunctional Betaine aldehyde dehydrogenase 2 (BADH2) enzyme, proving that mutations are not always hazardous, but a nature's gift. From an extensive literature survey and DNA sequence analysis based on sequences available in the Rice SNP-Seek-Database of the International Rice Research Institute, we identified different types of mutant alleles in the Badh2 gene that cause fragrance in rice. Of all mutation types, the badh2.1 allele, possessing an 8 bp deletion and 3 SNPs in the 7th exon of the Badh2 gene, was the most prevalent type across Asian rice germplasm in silico tested.
Accordingly, the second most prevalent was the badh2.7 allele, having a G insertion in the 14th exon. Multiplex PCR reported with ESP, IFAP, INSP and EAP primers well discriminates the badh2.1 allele from the non-fragrant allele. We developed a DNA marker, namely, Bad2.7CAPS to discriminate wild and mutant alleles of badh2.7, which could be used in marker-assisted selection (MAS) breeding studies. Similarly few such different types of markers have been reported by a few other countries, as they possess different rice varieties with varied types of mutations in the Badh2 gene, facilitating breeding varieties for rice fragrance. With the development of gene editing technology, several research papers have reported on the creation of novel fragrant alleles in the Badh2 gene of non-fragrant rice varieties. Accordingly, the Badh2 gene has been edited using the CRISPR/Cas9 tool, and finally, only Badh2-edited plants lacking the CRISPR/Cas9–Agrobacterium gene construct were selected by subsequent breeding. The volatile profiles have been confirmed with an increased level of 2AP in the badh2 gene-edited plants. Hence CRISPR/Cas9 tool has demonstrated as a promising method to introduce aroma trait to non-aromatic rice varieties by creating novel mutant alleles of badh2, which shortens the time taken for developing aromatic rice compared to cross-breeding followed by MAS methods.
Keywords: Fragrance, rice, Badh2, CRISPR/Cas9, 2-acetyl-1-pyrroline
