Identification Of Rice Chromosome Segment Substitution Line Z519 Carrying Major Purple Sheath Gene And PSH1 Mapping

With the improvement of people’s living standards, food production is higher for people’s demand, the quality of food are also increasing. Anthocyanin phenotypes, with strong visual detection, as a kind of natural plant pigment is enjoyed by people and has an extremely important role on food processing in our daily life. Similarly, anthocyanins in the hybrid purity identification, the transient expression of the target gene, as well as the trans-gene have a very important role in the instructions. Here, we identified a rice chromosome segment substitution line(CSSL) with purple sheath named Z519. Z519 was derived from advanced backcrosses between the recipient Nipponbare and donor R225 by SSR marker-assisted selection. In the study, the substitution segments of Z519 is identified, and comprehensive analyses of the Z519 are done by morphology, physiology and the important agronomic traits. Then, F2 population from cross of Nipponbare and Z519 is used for genetic analysis and gene mapping of the purple sheath. Finally the candidate gene prediction and sequencing, and subcellular localization of candidate genes, candidate gene expression pattern analysis are conducted.The main results were as follows:1. The identification of substitution segments of CSSL Z519Based on the preliminary studies, 82 SSR markers located on the substitution, together with 36 markers outside of the substitution segments are used to identify chromosome substitution fragments and detect genetic background using 10 individuals of Z519. The results showed that the substitution segments of 10 individuals of Z519 are consistent no residual segments of donor are detected, which indicated that Z519 is homozygous. Z519 carries 16 substitution segments from R225 whose total length is about 109.62 Mb and average length is 6.85 Mb. These substitution segments are distributed on 11 chromosomes except chromosome 10.2. Phenotype analysis and evaluation of main agronomic traits of Z519The bud sheath of Z519 begin to appear the purple color stripes when bud sheath is nearly 3 mm long. With the growth of rice plants, the purple stripes are displayed on leaf sheath, the edge of the stigmas, the vascular bundles of stem and leaves. While all parts of Nipponbare are green. 1000-grains weight of Z519 is significantly larger than that of Nipponbare. There are no difference between Z519 and Nipponbare in other agronomic traits, such as plant height, panicle number per plants, main panicle length, spikeletes number of main panicle, grains number of main panicle, and seed setting ratio. Thus, Z519 has important breeding value in molecular pyramiding breeding.3. Analysis of contents of photosynthetic pigments and anthocyanin of Z519Contents of Chlorophyll and carotenoid and anthocyanin of all the plants on the ground at seedling stage, together with them of leaves and leaf sheaths at heading stage were measured respectively between Z519 and Nippondare. The results shows that contents of the chlorophyll a and chlorophyll b and total chlorophyll at seedling stage are significantly lower than that of Nipponbare, and no difference is in carotenoid content. There are no difference between all contents of photosynthetic pigments in leaves of Z519 and Nipponbare at heading stage. Only contents of carotenoid exists difference in leaf sheaths between Z519 and Nipponbare. While there are no differences in contents of the chlorophyll a and chlorophyll b and total chlorophyll of leaf sheaths between Z519 and Nipponbare at heading stage. The the anthocyanin contents of the leaf sheaths are significantly higher than that in the Nippondare. While those of leaves display no difference between Z519 and Nipponbare at heading stage.4. Genetic analysis and molecular mapping of purple sheath in Z519F1 and F2 derived from cross of Nippondare and Z519 were used as populations of genetic analysis and gene mapping. All the leaf sheaths of F1 individuals are the purple. And apparent separation with green and purple sheaths is displayed in F2 progenies whose ratio of the purple sheath(724) to the green sheath(276) fitted completely to 3: 1 single dominant gene. Finally, the PSH1 was mapped on the chromosome 1 between In/Del marker LO3 and SSR maker LO1 with the physical distance of 37.8Kb.5. Candidate gene prediction and sequencing of PSH1According to the Gramene gene annotation information on the website, there are 6 genes altogether in the region, By sequencing two possible genes, a gene encoding plastid ATP/ADP transporter protein(LOC_Os01g45910) is found existing difference between Z519 and Niponbare. Three additional nucleotide(GTG) is inserted after the 255 th nucleotide in Z519 compared with Niponbare, which caused an amino acid(Gly) is added. Based on the above results, we preliminary identified LOC_Os01g45910 as the candidate gene.6. The expression pattern analysis of the candidate gene of PSH1The expression level analysis of candidate gene of PSH1 at seedling stage and heading stage was done by Real-time PCR. The results indicates that the expression level of candidate gene of PSH1 in leaf sheaths of Z519 is highest, and then is in leaves. No express is in root at seedling stage. At heading stage, the expression of candidate gene of PSH1 are detected in many organs of Z519 except roots and young panicles. The expression level is highest in the second leaf sheath from the top, then is in flag leaf, the first leaf sheaths from the top, the second leaf from the top, and stem.7. Expression analysis of genes related to the pigments metabolismReal-time PCR analysis of genes related to the photosynthetic pigments synthesis showed that the expression levels of all these genes associated with Chlorophyll, carotenoid metabolic and chloroplast development, plasmid encoding were lower in Z519 than that in Nippondare.8. The subcellular localization of the protein encoded by the candidate gene of PSH1A carrier subcellular localization of the candidate gene of PSH1 was constructed, and then was transformationed in rice protoplast. The results shows that the protein encoded by the candidate gene of PSH1 is localized in the chloroplast.