Identification And Gene Mapping Of A Narrow And Upper-albino Leaf Mutant Nul1(T) In Rice(Oryza Sativa L.)

Leaf blade is a main organ of photosynthesis and responsible for over ninety percent of rice yield. Therefore, increasing utilization efficiency of light energy is significant in crop breeding. In general, leaf color represents the content of photosynthetic pigments and is directly related to photosynthesis. In rice production, suitable narrow and roll leaf blade could improve plant phenotype of crop group and then facilitate acception and application of light energy. Therefore, studing leaf blade development is valuable and significant in rice ideal plant type breeding. Up to date, at least four genes, NAL1, NAL2, NAL3, NAL7 and NAL9, have been cloned by map-based strategy and proved to regulate narrow leaf characteristics. Except for the NAL9, other genes were all related with IAA biosynthesis or signal transduction. In this study, a spontaneous mutant has been discovered from No. B78 in the paddy field. The mutant displayed narrow leaves with albino in adaxial surface just like EMS (ethyl methane sulfonate)-induced mutant null reported by Wang et al. (2012). Given this reason, the novel mutant was named as narrow and upper-albino leaf 1, temporarily null(t). This paper performed systematic studies in morphological characteristics detection, identification of photosynthetic pigments and characteristics, cytology observation, genetic analysis, gene fine mapping and candidate gene identification etc.The main results were as follows:1. Morphological analysis and agronomic traitIn the paddy field, the null(t) displayed narrow and upper-albino leaf blades throughout the development. At the seedling stage, leaf width of the first, second and third respectively occupied 50.11%,50.90% and 62.13% of the wild type, the declining led to significant different at the 0.01 level while leaf lengths have no significant changing. Compared with those of the wild type, agronomic traits were influenced in the mutant. That is, effective panicle number per plant increased significantly, seed width and length, seed setting rate and 1000-grain weight decreased significantly, plant height and number of grain per panicle have no changing.2. Photosynthetic pigment and characteristicsCompared with the wild type, the mutant has similar contents of photosynthetic pigments in the flowering stage. That is, except for chlorophyll a in the flag leaf, the contents of chlorophyll a, chlorophyll b and carotenoid have no significant differences in three functional leaves. There were lower photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr) and water use efficiency (WUE) in the null(t) than those of the wild type, however, only Gs decreasing led to significant level. And this result was corresponding to photosynthetic pigment contents.3. Cytology observation in the leaf bladeThe flag leaf of null(t) has 7.17 large veins and 34 smaller veins, which were fewer than those of the wild type. The number of smaller veins changes nothing between the adjacent large veins while the reduced distance between adjacent small veins was detected, only occupied 69.54% of the wild type. Further analysis showed that there were eleven cell layers between adjacent small veins in the wild type and which were only seven layers in the null(t), this should be the main reason of null(t) narrower leaves. In the thinner position of mesophyll, seven layer cells were observed in the mutant while only five layers appeared in the wild type. This should be responsible for the influence of photosynthetic pigment.4. Genetic analysis of the null(t)Jinhui 10 was crossed with the null(t) for genetic analysis. The shape and color of leaf blade displayed normal in the F1 progeny. In the F2 progenies, the segregation ratio of normal plants (2458) to mutational plants (806) has no significant difference with the ratio of 3:1(x2=0.16<x2 0.05=3.84). Meanwhile, the F2 plant showed narrower leaf blade and also displayed albino in the adaxial surface, just like null(t). Given these, we predicted that the mutational characteristics of null(t) should be regulated by one nuclear recessive gene and the gene must be responsible for both narrow and albino leaf traits.5. Fine mapping of NUL1(t) geneBased on 806 F2 recessive individuals from nul1(t)/Jinhui10, the NUL1(t) gene was finally mapped on chromosome 7 between Indel marker Indel 07-1 and Indel 07-5 with 51.5 kb physical distance. According to Rice Genome Annotation Project (RGAP), four annotated genes were discovered in the region and they encoded CHR4/MI-2-LIKE, peptide-nasparagine amidase, MYB family transcription factor and express protein, respectively.6. Analysis of candidate genesAnnotated genes were sequenced in the region and a mutation in the 2396th base of LOC_Os07g31450 coding sequence was detected, the gene encoding CHR4/MI-2-LIKE protein, it was G in the wild type and was A in the nul1(t), this caused amino acid changing from serine to asparagines. Therefore, LOC_Os07g31450 was predicted as the candidate of NUL1(t).7. Identification of NUL1(t)Four novel mutants displaying similar phenotype of narrow and upper-albino leaf blades were discovered from the maintainer line Xida 1B with seeds treated by EMS. Crossing with nul1(t) respectively, the phenotype of F1 generations from two novel mutants nul1(t)-2 and nul1(t)-3 showed both narrow and albino leaf blade, just like nul1(t), and the F2 generations have no separation. Sequencing showed that nucleotide A at the 3039th of LOC_Os07g31450 was deleted in the nul1(t)-3 compared with those of the wild type. Therefore, the gene encoding CHR4/MI-2-LIKE protein was proofed as the gene of NUL1(t).