Fine Mapping Of A Narrow And Rolled Leaf 2 (NRL2_(t)) Gene In Rice (Oryza Sativa L.)

Leaf is the main photosynthetic organ, which is an integrant element of plant architecture. In super-high yield breeding of rice, the researchers found that moderate rolling leaf could keep leaves erected, optimize canopy light transmission and improve the photosynthesis rate of the upper leaves, which could increase the rice yield and grain quality. In this study, a rolled leaf mutant (nrl2(t)) with reduced leaf width was obtained from Oryza Sativa subsp. Indica line, Jinhui10 which was treated by ethylmethane sulfonate (EMS). We reported herein the genetic analysis and the morphological characteristics of the mutant and fine mapping of the gene NRL2(t), which could lay a good foundation for gene cloning and molecular mechanism study.1 Agronomic trait and morphological analysisFrom the 4th leaf stage, the nrl2(t) mutant exhibited narrow/rolled and elongate leaf, earlier flowering, thinner stalk. Furthermore, the seed setting rate, the first branch number and the grain number per panicle were all reduced significantly. The I2-KI staining showed that there are three different kinds of pollen granule in the mutant. They were not-fully staining pollen granule; pollen granule with abnormal size, shape and starch content and normal pollen granule in the mutant. It was found that the shape, size and arrangement of the bulliform cells which were at the joint between midrib and blade had changed a lot. Besides, the number of lateral veins had reduced in both the large vascular bundle and the small ones of the nrl2(t) mutant per blade. And the similar variation happened in the stem tissue. All of these implied the mutant of NRL2(t) has caused multiple morphology change.2 Chlorophyll, IAA and CTK content analysisThe indole-3-acetic acid (IAA) and cytokinin (CTK) content had relatively reduced in the flag leaf compared to those of wild type, but increased in the young panicle at the heading stage, particular the CTK content in the young panicle. Besides, the pigment contents in the nrl2(t) mutant were higher than those of the control.3 Genetic analysisThe nrl2(t) mutant successively exhibited rolling and narrow leaves for 4 generations in Chongqing and Hainan, China. This outcome indicated that the mutation trait was stably inherited. In the F1 generation which derived from Xinong 1A×nrl2(t), all the hybrids displayed normal leaves which suggested that the narrow and rolled trait was controlled by a recessive gene. In the F2 segregating population, the ratio of narrow and rolled leaves to normal leaves was 1025:3207, which was ideally fitting the 1:3 ratio (χ2=1.33<3.84[χ2(0.05.1)]).All of these results suggested that the narrow and rolled leaf phenotype of the nrl2(t) mutant was controlled by a single recessive nuclear gene.4 Gene mappingBased on the BSA method,83 SSR markers were found polymorphic between Xinong1A and nrl2(,) after the parental screening for a number of 420 SSR markers which were applied for the genome mapping. NRL2(t) gene was located between RM1284 and RM5748 on chromosome 3, with a genetic distance 4.78 and 6.50 cM respectively. Within the region between RM1284 and RM5748,26 new SSR markers and STS markers were developed. After that, NRL2(t) gene was located between SSR markers s3RM1 and s3RM3, with genetic distance of 0.07 and 0.1 cM respectively. And NRL2(t) was co-segregated with SSR marker s3RM2.5 Real-time PCR analysis of 5 reported genes related to narrow and rolled leaf in mutantReal-time PCR analysis was conducted in this study in order to quantify the expression of several genes which are related to narrow and rolled leaf trait. And the results revealed that OsAGO7, NAL1, NAL7 and SLL1 were down-regulated in the mutant. On the contrary, the NRL1 which was involved in the biosynthesis of cell-wall polymers was up-regulated a lot.