Studies on longevity in rice seeds focusing on the stability of embryonic RNAs

概要

The seed longevity is an important agronomic trait and to maintain the germinability for long time is strongly demanded in the seed industry. It has been shown that RNA stability in the embryos of the dry seeds is related to seed longevity in various crops. However, the mechanism of decline in germinability mediated by degradation of embryonic RNAs remains unidentified. Rice, one of the model plants, has a lot of useful characteristics to understand the molecular mechanisms of various phenomena. In this study, the seed longevity of rice was analyzed focusing on the stability of embryonic RNAs. The objectives of this study are i) to analyze the correlation between seed longevity and RNA integrity in embryos; ii) to find out the best cultivar(s) as potential breeding materials with high germinability, long and stable seed longevity trait; and iii) to identify the genes responsible for the seed longevity of rice cultivar.

In chapter 1, the relationship between seed longevity and the integrity of RNAs in embryos from the japonica rice cultivars was examined. Generally, many years would be needed to evaluate seed germinability under natural storage conditions. To circumvent this problem, an artificial aging technique known as controlled deterioration treatment (CDT) has been widely applied to accelerate the ageing process with high temperature and relative humidity to easily and quickly estimate seed viability. Therefore, seed samples were subjected to CDT and the total RNAs were extracted from the embryos. These RNAs were analyzed by electrophoresis using an Agilent 2100 Bioanalyzer system (Agilent Technologies), and then RNA integrity number (RIN), which evaluates RNA fragment size distribution, were calculated using Agilent 2100 Bioanalyzer software. Degradation of RNA from embryos of a japonica rice cultivar “Nipponbare” was induced by CDT before the decline of the germination rate and the positive relationship between seed germinability and integrity of embryonic RNAs was observed. Moreover, this relationship was confirmed in the experiments using aged seeds from the “Nipponbare”, “Sasanishiki” and “Koshihikari” rice cultivars. In addition, the RNA integrity number (RIN) values, calculated using electrophoresis data and Agilent Bioanalyzer software, had a positive correlation with germinability (R2 = 0.75).

In chapter 2, the screening was performed on germinability of rice seeds after “CDT” or “long-time naturally ageing” treatments using core collection, Rice Diversity Research Set of germplasm (RDRS) established by the National Agriculture and Food Research Organization (NARO) in Japan in order to obtain the rice cultivar(s) having long and stable seed longevity trait as useful potential breeding materials. This collection is a set of world rice cultivars developed from about 300 accessions of rice selected according to their origin information, and determined 69 cultivars based on a restriction fragment length polymorphism (RFLP). Among all, the longevity in seeds of three cultivars, such as “Vandaran (indica)”, “Tupa 729 (japonica)” and “Badari Dhan (indica)”, were significantly and constantly higher under CDT and long periods of storage comparing with other varieties. Suggesting that these cultivars can be used as useful potential breeding materials for improvement in seed longevity. Also, the positive correlation between the seed longevity and RIN value was confirmed in this experiment using seeds of RDRS.

In chapter 3, the genetical analysis was carried out to identify the gene(s) responsible for long longevity in rice seeds. Previously, it was reported that the seed longevity of “Habataki” (inica) cultivar is longer than that of “Sasanishiki” (japonica) cultivar. Therefore, the chromosome segment substitution lines (CSSLs) derived from “Sasanishiki” and “Habataki” were used for this analysis as materials. Based on the data of genotyping of CSSLs and their progenies, the gene(s) responsible for the seed longevity of “Habataki” is located on chromosome 2 at the position between 32.8 – 42.1 cM regions. Moreover, it has been suggested that the product(s) of the gene(s) responsible for seed longevity on chromosome 2 is/are involved in keeping the integrity of embryonic RNAs under aging condition of long-term storage at 4°C and artificially treatment of CDT. Subsequently, a significant and positive correlation (R2 = 0.96) between germinability and RIN values was obtained in the experiments of this chapter. Taken together, it was concluded that the stability of embryonic RNAs is involved in maintaining seed longevity over time and RIN values can serve as a standard index to evaluate germinability in rice.