Genetic Analysis And Fine-mapping Of The Male Sterile Mutant D63 And Yellow Leaf Mutant 577ys In Rice

Male sterile and leaf color mutants are common types of mutation, and are also important materials for rice breeding and functional genomes research. In this study, we have obtained the male sterile mutant D63 and the yellow leaf mutant 577ys from indica variety G46B and japonica variety Nipponbare, respectively, through ethyl methane sulphonate (EMS) chemical mutagenesis. Subsequently, we carried out phenotypic investigation, genetic analysis and gene mapping of the two mutants. The main results are as follows:1. Phenotypic characteristics and major agronomic traits of the male sterile mutant D63:In the D63 mutant, the female organs normally develop, and number of the male organs (anthers) is also normal six pieces. But its anthers are small, thinner and milk white, and there is no pollen in its pollen sac. The male sterile trait of D63 is not influenced by light and temperature conditions, so belongs to common nuclear sterility type. During the mature period, the plant height and panicle exsertion of D63 reduced by 13.7% and 266.7%, respectively, compared with those of the wide-type parent G46B. The other major agronomic traits, such as number of productive panicle per plant, panicle length and 1000-grain weight, were not significantly affected by its male sterility.2. Genetic analysis of male sterile trait of the D63 mutant:Genetic analysis showed all F1 plants displayed normally fertile phenotype, and the number of fertile plants versus sterile plants in the F2 populations was fitted to 3:1, indicating that the D63 sterile phenotype was controlled by a single recessive nuclear gene.3. Gene mapping and candidate gene analysis of the male sterile mutant D63:The mutant gene was mapped to the region between InDel markers J2 and J4 and near SSR marker RM300 on rice chromosome 2. The genetic distances from the sterile gene to the two InDel markers were 0.2 cM and 0.1 cM, respectively, and the physical length of this region was 105.8 kb, in which 16 putative genes were annotated in the Rice Genome Annotation Project. Sequencing analysis of candidate genes between the mutant and its wild-type revealed a single-nucleotide A-to-C mutation existed in the D63 mutant at position 1580 in coding region of the LOC_Os02g28970 gene for secretory fasciclin glycoprotein, which resulted in an amino acid change from His at position 527 to Pro in the encoded product.4. Phenotypic characteristics and major agronomic traits of the leaf colors mutant 577ys:The mutant displays yellow leaf throughout growth period, and its leaf has many rusty spots at seedling stage, which is obviously different from the wide-type parent Nipponbare. During the mature period, plant height, number of productive panicle per plant, flag leaf length, seed setting rate,1000-grian weight of 577ys reduced by 15.2%,30.4%,8.0%, 9.0%,10.2%, respectively, compared with those of the wide type. Additionally, growth period of the mutant was delayed by about 14 day. The other agronomic traits were not significantly affected by its yellow leaf mutation.5. Photosynthetic pigment contents of the yellow leaf mutant 577ys:Compared with the wide-type parent, during seedling stage, Chi a, Chl b and carotenoids of 577ys reduced by 81.34%,92.75%, and 57.89%, respectively. During heading stage, its Chl a, Chl b and Carotenoids reduced by 49.56%,70.42%, and 48.6%, respectively. This result reveals that photosynthetic pigment synthesis in the leaf color mutant was limited.6. Chloroplast structure of the yellow leaf mutant:Observation under transmission electron microscope suggested that the chloroplast thylakoids was damaged, number of grana lamella reduced and its arrangement was not regular in the 577ys mutant, compared with those in the wide-type parent. Furthermore, number of osmiophilic plastoglobuli obviously increase.7. Gene mapping and candidate gene analysis of the yellow leaf mutant 577ys:The mutant gene was firstly mapped to the region between SSR marker RM129 and RM259 on rice chromosome 1. Furthermore, we have designed several InDel markers, which mapped the mutant gene to a region between InDel marker Zl and SSR marker RM572. The genetic distance from the mutant gene to the two markers is 0.3cM and 0.1 cM, respectively, and the physical length of this region was 105.4 kb, in which 13 putative genes were annotated in the Rice Genome Annotation Project. These putative genes are being sequenced to find the candidate gene for 577ys mutation.