Fine Mapping And Isolation Of Candidate Genes Involved In Rice Fragrance

Fragrance is one of the most important traits in rice. From more than 100 volatile flavor components, 2-acetyl-1-pyrroline (2AP) stands out as the key component of aroma in both basmati- and jasmine-style fragrant rice. It has been reported that a single recessive gene (fgr) on chromosome 8 is responsible for the production of 2AP.In our study, three crosses were made between indica and japonica rice varieties with the contrasting traits, including Wuxiangxian/Zhongai91, Wuxiangjing/Nanjing11 , and Suyunuo/Zhongxian3037 , from which segregant populations (BC1, F2, and F3) consisting of 3280 individulas were derived by both backcrossing and self-crossing.In the process of fragrant assay, fresh leaf tissue at the tillering stage was chopped into small pieces and immersed into 1.7% KOH in a petri dish. After 10 minutes, the dish was opened, and volatile flavor emitted from leaf tissue could be immediately smelled. Alternatively, tasting individual seeds was also a favorable way to detect fragrance. For some important recombinants, their fragrant phenotypes were accurately determined by evaluating their offsprings.Our initial mapping efforts with SSR markers confirmed the previous mapping result, and placed the fgr gene into a region flanked by RM8264 and RM1109. In thefine-mapping process, the fgr region was saturated with high-density markers developed by exploiting sequence diversities between indica and japonica rice subspecies. After mapping with segregant populations consisting of totally 3280 individuals, the fgr locus was restricted to an interval of 408kb flanked by the left marker K10 and the right marker M10. Within the K10/M10 interval, the recombinants were further resolved to delimit the fgr locus into a 69kb DNA segment. The candidate fgr genes were identified after searching for annotated genome sequence of the japonica rice cv. 'Nipponbare'. With an aim to clone the fgr gene, two rice BAC libraries, constructed from a fragrant cv. 'Suyunuo' and a non-fragrant cv. 'Nanjing 11', were screened with closely linked markers, resulting in the positive clones encompassing the fgr locus. With these BAC clones, all fgr candidate genes have been cloned into express vector for functional testing.