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ARS Home » Southeast Area » Stuttgart, Arkansas » Dale Bumpers National Rice Research Center » Research » Publications at this Location » Publication #282402
Title: Cytoplasmic effects on DNA methylation between male sterile line and its maintainer in rice

Author
item PIZHOU, XU - Sichuan University
item Yan, Wengui
item HONGYN, ZHANG - Sichuan University
item LIMENG, JIA - Zhejiang University
item XUHAO, PAN - Sichuan University
item XIANJUN, WN - Sichuan University

Submitted to: Rice Technical Working Group Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 2/27/2012
Publication Date: 2/1/2013
Citation: Pizhou, X., Yan, W., Hongyn, Z., Limeng, J., Xuhao, P., Xianjun, W. 2013. Cytoplasmic effects on DNA methylation between male sterile line and its maintainer in rice. Rice Technical Working Group Meeting Proceedings. Feb. 27-Mar. 1, 2012. Hot Springs, AR. pg. 58.

Interpretive Summary:

Technical Abstract: Hybrid rice is advantageous over the traditional one on food production, which is important to support the increasing world’s population, especially in the developing countries. Three-line system that has played a major role since the 1970s in rice includes male sterile (A line), its maintainer (B line) and restoring (R line). A and B lines share same nucleus, but have different cytoplasm which is male sterile for the A and fertile for the B. Therefore, any differences between A and B lines are due to cytoplasm. Besides pollen sterility, A line heads 3-4 days later, exserts fewer portions of panicles, flowers less concentrated, and is shorter in height than B line. Microarray analysis revealed a variation of expression levels for a large number of nuclear genes between A and B lines, indicating cytoplasmic effects on nuclear gene expression. Recently, differences of cytosine methylation in DNA between reciprocal hybrids have been reported in rice. DNA methylation is known to play an important role on regulating gene expression in eukaryotes. Inspired by these reports, our objective for this study was to characterize cytoplasmic effects on cytosine methylation using popular A and B lines in Chinese hybrid rice production. Four different A lines (Jin23A, G46A, D62A and II-32A) representing four major cytoplasmic male sterile (CMS) types (WA-type, G-type, D-type andYinshui-type), respectively and their corresponding B lines (Jin23B, G46B, D62B and II-32B) were comparatively studied. Genomic DNA was extracted from same organ tissues of the experimental materials in seedling stage. Methylation-sensitive amplified polymorphism (MSAP) technique was applied to DNA digestion using two restricted enzyme combinations, EcoR I+Msp I and EcoR I+Hpa II, separately. Twelve pairs of primers were selected to detect cytosine methylation at 5’-CCGG site after electrophoresis. The methylation was indicated by either presence or absence of fragments in the gel. Among the four pairs of rice lines, a total of 350 fragments were amplified. The degrees of DNA cytosine methylation ranged from 16.56% to 20.43% among the eight lines. All the A lines had lower cytosine-methylated degree than their corresponding B lines with the most difference of 3.05% for WA-type (Jin23A - 17.38 and Jin23B - 20.43), followed by 2.40% for Yinshui-type (II-32A - 17.96 and II-32B – 20.36), 1.23% for D-type (D62A - 18.94 and D62B – 20.17) and 0.62% for G-type (G46A – 16.56 and G46B – 17.18). A and B lines are polymorphic for a type of CMS if DNA methylation happens in only one of the lines for a fragment or restricted site. There were 20 polymorphic sites between the A line and B lines in WA-type and Yinshui-type, but only 5 polymorphic sites in G-type and D-type. Indicated by the above results, we reached the following conclusions: 1) rice cytoplasm affected DNA methylation in the nuclear genome and male sterile cytoplasm decreased the degree of cytosine methylation in comparison with fertile cytoplasm, 2) decrease levels classified the four male sterile cytoplasm in two groups, high (WA-type and Yinshui-type) and low (G-type and D-type), and 3) the decrease level was positively associated with the number of polymorphic sites of DNA methylation. Thus, the number of polymorphic sites should be another estimator on cytoplasmic effect. Previous study has shown that methylation polymorphism could also be taken as a useful tool to detect the genetic distance. Gambiaka Kokoum and Dissi D52/37, indica cultivars in Mali of West Africa, are the donors of male sterile cytoplasm to G-type and D-type, respectively. The donor to WA-type is a male sterile plant of common wild rice Oryza sativa L.f. spontanea. Yinshui-type’s donor used to be a cultivar in Indonesia, Shui Tian Gu 6. Their donors for fertile cytoplasm and nucleus are all indica breeding lines in China. Therefore, the differenc