Identification And Gene Mapping Of A Late Rolled Leaf Mutant Lrl1 In Rice(Oryza Sativa L.)

Rice is one of the most important crops all over the world. Improving rice yield is important to solve people s problem of food. Rice leaf is the main photosynthetic organ. And leaf shape is an important part for plant type breeding. Studies indicated that moderate rolling leaf could keep plants upright, increasing transmittance in the lower leaf, then improving the light condition of population, which laid a good foundation for increasing rice yield. However, earlier and over-rolling of the leaves may be difficult to reach ideal effect due to influencing plant growing and development. Here, we obtained a late moderate-rolled leaf mutant lrll in rice deriving from the restorer line Jinhui10 (Oryza sativa L. ssp. indica) treated by EMS. In the study, the mutant lrll was identified by morphology, physiology, cytology. Furthermore, a series of the studies about the gene IRL1 were done, such as genetic analysis, gene mapping, candidate gene prediction and sequencing, and QPCR analysis of cloned gene for rolled leaf in lrll and the wild type rice. The main results were as follows:1. Phenotype and the primary agronomical characters of the lrll mutantThe leaves of lrll were normal in early development stages. Then, the upper 3 leaves began rolling inward along the vein after the thirteen leaf age, and the rolling index increased with the plant development. The rolling index of 1st,2nd, and 3rd leaf from the top in lrll was 60.83%,58.63% and 20.70% at booting stage, and73.66%、 66.91% and 45.81% at maturity stage. The width of 1st leaf from the top in lrll was significantly narrowed than that of wild type. However, there were no difference between the width of other leaves in lrll and J10. Compared to the wild-type, most of the important agronomic traits in lrll existed no significant difference, such as plant height, number of skikeletes per panicle, number of grains per panicle, seed set rating, number per panicle and panicle length. Only 1000-grains weight were significantly less than that of the wild type, which indicated that the lrll has great values in breeding because it has many favorable agronomic traits for breeding besides rolling leaf.2. Cytology analysis of the lrll muatntIn order to reveal the reason of leaf rolling of the lrll. The paraffin section of both the leaves of the wild type JinhuilO and the mutant lrll were made, and observed under microscope. The results showed that the number and shape of bulliform cells between the adjacent small vascular bunbles of the lrll leaf blade existed difference compared with that of the wild type J10. In mutant lrll, the number of bulliform cells decreased, and the shape of them arranged Irregularly and become more larger. These changes of bulliform cells made the angle among small vascular bundles become smaller and then maybe cause the leaf rolling.3. Physiology analysis of the lrll mutantAt the heading stage, chlorophyll and carotenoid content in the lrll and the wild type J10 were measured. The results showed that the chlorophyll a and chlorophyll b, total chlorophyll and carotenoid content were all significantly higher than that of wild type, indicating that there were better photosynthetic bases in lrll than that of Jinhui10. Furthermore, the photosynthetic characteristics of wild type and mutant are analyzed and found that the net photosynthetic rate, stomatal conductance, transpiration rate in the lrll exsisted no difference with those in wild type.4. Genetic analysis of the lrll mutantSterile line Xiongnong 1A with flat leaf was crossed with the mutant lrll for genetic analysis. The leaves of all F1 exhibited flat leaf as Xiongnong 1 A, suggesting that the late rolled leaf was controlled by recessive gene. Besides, There was an apparent separation in F2 progenies, in which normal plants were 3005 and mutant plants 960. By x2 test, the ratio of normal to mutant plants fited completely to 3:1 (x2=1.30<<x20.05,1=3.84), indicating that the rolled leaf mutant was controlled by a single recessive nuclear gene.5. Gene mapping, candidate gene prediction and sequencing of partial possible candidate gene480 SSR markers distributed on 12 chromosome of rice were firstly used to screen the polymorphism between Xinong 1A and lrll. And 98 markers were found existing polymorphism. The 98 markers were then used to analyze linkage between two gene balks of rolled leaf and normal leaves. The maker RM201 on chromosome 9 were showed linkage with the mutation gene. Then, the target gene was initial positioning between Indl and RM201 with 2.4 cM and 4.0 cM. By further designing new markers, LRL1 was finally mapped between SWU-1 and Ind 6, the physical distance is 812 kb. By candidate gene prediction in the area, there are 129 genes altogether. Through sequencing, three genes possiblly related to rolled leaf were found existing no difference between lrll and J10.6. Real-time PCR analysisIn order to find the clue of rolled leaf genes. Real-time PCR analysis were used to reveal the relationship among the LRL1 and genes for rolled leaf related to bulliform cells changes. In the leaves of the lrll mutant, the expression quantity of ACL1, SRL1 and NAL7 were down-regulated significantly, that of ROC5 and RL14 were up-regulated significantly. While that of the gene NRL1 was very low in both leaves of wild type and lrll mutant, indicating that the genes might be not expressed in the leaves.