Genetic Identification And Candidate Gene Analysis Of Narrow Leaf And High Tillering Mutant Lt1 And Yellow-green Leaf Mutant 7066ys In Rice

The leaf color, leaf type and tillering are important traits to rice yield, the leaf color mutants have brought to favour by breeders for rice functional genomics research, and leaf type and tillering mutants are important material for ideal plant type breeding. Therefore, the study of these mutants can provide theoretical and material for high yield breeding.In this study, based on chemical mutagenesis (EMS), we had obtained three narrow leaf and high tillerings mutant lt1-1, It1-2, It1-3 (japonica rice variety Nipponbare), and a yellow-green leaf mutant,7066ys, from the mutant library of japonica rice variety Zhonghua 11 of dense and erect mutant dep2. Furthermore, we carried out the genetic analysis, phenotypic observation and main agronomic traits investigation of lt1 and 7066ys. The results were as following:1. Genetic identification and candidate gene analysis of narrow leaf and high tillering mutant It1 in riceGenetic analysis indicated that lt1 was controlled by a single recessive nuclear gene. Using the F2 population of lt1/G46B as mapping population, the target mutant gene was mapped in a region of 64.4 kb between two InDel markers S3 and S4 on the long arm of chromosome 4, and the genetic distance were 0.66 cM and 0.78 cM, respectively, Predicted genes of sequencing analysis revealed that a single base was changed (G1102A) in the coding region of the LOC_Os04g52479 gene which encodes a plant-specific protein, causing a missense mutation (D368N).In addition, we further screened other the two allelic mutants lt1-2 and lt1-3, sequencing analysis of lt1-2 revealed that a single base was changed (C1201T) in the coding region of the LOC_Os04g52479 gene, causing a missense mutation (P401S), and lt1-3 deleted a 26 bp fragment (from 206 bp to 231 bp) in the coding region of the LOC_Os04g52479, causing frameshift mutations, codon early termination. Compared with the wild type, three allelic mutants are short by 52.8%,52.5%,70.4% in blade leaf length, respectively, and more than 60% reduction in leaf width. Comparison of the internode elongation patterns between It1 and wild-type revealed that the corresponding internodes from top to bottom (designated internodes ?, ?,?,?, and ?, respectively) of the lt1-1 mutation were decreased by 31.9%,34.9%,51.7%,41.6% and 63.1%, respectively; lt1-2 was similar to lt1-1; and lt1-3 was more serious than lt1-1 and lt1-2 except the fifth internodes. At maturity, compared with the wild type, lt1-1, lt1-2 and lt1-3 tillers were increased by 112.6%,79.3% and 348.3%, respectively. This study showed that lt1-3 most amino acids deletion is main reason for serious mutant phenotype than other allelic mutant in plant height and tillers.The results show that LT1 was a new allelic to NAL1 which had been reported before, but the phenotypes of each lt1 and nal1 mutant was not completely consistent, this might be due to the differencesof materials background and mutant sites. This study could help to figure outthe regulatory mechanism of rice leaf type and tillering for further studies in ideal plant architecture of rice.2. Genetic identification and candidate gene analysis of yellow-gre-en leaf mutant 7066ys in riceBased on main agronomic traits investigation, we found that the 7066ys mutant exhibited yellow-green leaf phenotype throughout the growth period. Compared with the wild type,7066ys exhibited short plant height, grains per panicle and number of productive panicles per plant reduced by 17.7% and 19.7%, respectively, other agronomic traits such as panicle length, seed setting rate and 1000-grain weight were little different. Compared the chlorophyll content of 7066ys and the wild type in seedling stage and booting stages, the chlorophyll a of 7066ys decreased by 72.3% and 54.4%, respectively, in 7066ys; Chlorophyll b of 7066ys decreased by 94.8% and 92.9%, respectively. By the transmission electron microscopy analysis of 7066ys and the wild type in seedling stage, the result revealed that the chloroplasts in the 7066ys mutant were out-of-shape, thylakoid lamellae decreased in 7066ys, the results show that 7066ys chlorophyll synthesis and chloroplast normal growth and development hindered.Genetic analysis indicated that 7066ys was affected by a single recessive nuclear gene. Using F2 population of 7066ys/M63 as mapping population, the mutant gene was mapped to a region of 3208 kb between two InDel markers D1 and D3 of chromosome 3, and the genetic distance were 0.31 cM and 0.16 cM, respectively, sequencing analysis revealed that a single base was changed (G790A) in the coding region of the LOC_Os03g36540 gene which encodes Chll subunits of Mg-chelatase, causing a missense mutation (G264R).This study showed that 7066ys is probably allelic to Chl9, but they had different phenotypes. The different phenotypes of the two mutants might be caused by the different mutational sites of 7066ys and Chl9 genomic sequence and genetic background. This study provides a theoretical basis for the mechanism of chlorophyll synthesis and the rice leaf color marker for cultivate high-yield rice breeding.