| Plastid development from proplastids to photosynthetically active chloroplasts is one of the most important metabolic processes during plant growth and is coordinately regulated by both plastid and nuclear genes. Chloroplast development is largely under nuclear control, because the coding capacity of plastids is very limited and nuclear genes encode more than 95% of the chloroplast proteins. Thus, the precise coordination of gene expression through two-way signaling between plastids and the nucleus is essential for chloroplast biogenesis in plant cells. Utilizing leaf color mutants to research synthesis of chlorophyll and chloroplast formation has important value in use.A green-revertible albino mutant was isolated by chemical mutagen EMS from a late maturity mutant 10079 of Nipponbare.The phenotypes and main agronomic traits of the mutant were observed, pigment contents in leaves of wild-type and gra75 at different stages were measured, microstructure of chloroplast was abserved of 10079 and gra75. Genetic mapping of the mutant gene was conducted by using molecular marker and F2 mapping population of gra75/Zhefu802, found the candidate gene LOC_Os06g07210.Phenotypic characteristics and agronomic traits of the mutant gra75:Under nature conditions, gra75 plants exhibit a normal green phenotype up to the L3 stage. Form the fourth leaf blade to the seventh leaf blade produce almostly fully albino leaves, but then they develop nearly normal green leaves after L8 stage, the albino leaves also reverting to normal green gradually. From booting until the maturity, gra75 mutant phenotype with no significant differences between wild-type. Plant height, panicles per plant, Panicle length, spikelets per panicle, seed setting rate,1000-grain weight and other agronomic traits were investigated at maturity stage, the results showed that the mutant and wild type were not significant.The measure of chlorophyll and carotenoid contents:The albino leaves (the fifth leaf) of the gra75 mutant had 88.3% and 88.5% reduction of Ch1 a and Ch1 b, respectively, and 79.3% reduction of carotenoid levels compared to those in wild type at Seedling stage, which showed that pigment synthesis severely disrupted at albino stage, indicating that the mutant phenotype resulted from reduced Ch1 level. After reverting to green, eventually, mutant plants accumulated substantial quantities of Ch1 and carotenoid, reaching almost the wild type levels and even slightly higher than the wild type at booting stage. Together this suggests that after reverting to green the level of pigment synthesis will be back to normal.The structure of mesophyll cell and chloroplast:The albino mutant mesophyll cell imperfect development and were found to contain undifferentiated chloroplasts that were fewer and smaller, the number of organelles decreased. About the internal chloroplast, both the number of osmiophilic particles is not significantly different, and were evenly distributed in the chloroplast stroma, but the number of thylakoid lamellae, grana and starch grains were significantly decreased in mutant chloroplast. Shows that the mutant development of chloroplast was seriously inhibited in albino leaves.Genetic analysis of the mutant character of gra75:F1 generations of gra75 crossed with the normal green varieties japonica cv. Nipponbare, indica cv. Zhefu802 and Minghui63 were all normal green. Individuals of the F2 generations could be classified into two groups with very obvious difference in color, i.e. normal green and albino. The chi-square (χ2) test, in which the segregation ratio fitted the expected ratio of 3:1. These results indicated that the green-revertible albino character of gra75 was controlled by one pair of recessive nuclear gene.Molecular marker mapping of the albino gene of gra75 and candidate gene analysis:To map the locus, an F2 mapping population was generated from a cross between the gra75 mutant with Zhefu802. Finally, gra75 gene was mapped between InDel marker HC1 and HC2, the genetic distances were 0.06 cM and 0.6 cM respectively and the physical distance was 120kb. Analysising and sequencing the candidate genes at the limited genomic region, found that the genomic structure of RNRL1 (ribonucleotide reductase large subunit) comprising 17 exons and 16 introns, a single nucleotide change (C716T)in exon 5 of the gra75 allele caused a missense mutation, Ala to Val. Therefore, the Os06g07210 gene was considered as the candidate gene of gra75. The same gene mutation caused by similar V3 (Virescent3) was documented previously. The two mutants traits incongruous may be due to the large subunit of ribonucleotide reductase (RNRL) different amino acid sites changed.The potential applications of GRA 75 gene:The green-revertible character at seeding stage of mutant gra75 could expressed steady, and that albino phenotype no Significant influence on main agronomic traits at mature stage. This result provides a foundation of map-based cloning of gra 75 gene as well as its molecular marker-assisted breeding. |
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