CHROMOSOME AND GENOTYPE CHARACTERIZATION IN SOME RICE BIOTYPES SHOWING GRAIN SHATTERING

Red rice (Oryza sativa var. silvatica L.) is a common weed in rice crop, very troublesome to control due to high shattering. The aim of the present research was to analyse the genome of some biotypes of red rice in order to promote innovatives methods for its control. In the first year, morphological and karyotypical analysis of some phenotypically different red rice biotypes in comparison with the rice cultivar ‘Loto’ were carried out. The seeds of rice and red rice were sown in greenhouse and afterwards the root tips of the seedlings were employed for the cytological analysis. The chromosome preparations were made by the standard air-drying technique, after enzymatic maceration, and stained with a 4% Giemsa solution for 20 min., according to Fukui and Iijima’s method (1992). The analysis, by means of a computerized chromosome image method (CHIA–EA), showed that in red rice various translocations occur, which always involve a chromosome of the first pair together with other elements of the set. Previous results clearly indicated that each red rice biotype is characterized by a specific translocation, showing a relationship among different phenotypes and karyotypes. It is important to note that in Oryza sativa one gene for shattering maps to the long arm of chromosome 1, which is always involved in the translocations. The project is divided into three phases: the field growing and observations, the laboratory analysis and the computer analysis. The first results that we have obtained support the proposed assumption. An increase in the dose of the genes frequently alters the phenotype, depending upon the effect of the genes involved. In the second year, the research was based on the molecular analysis. Since the shattering gene is located on the long arm of the chromosome 1, which is involved in the translocation process, I have analyzed this genomic locus by both cytogenetic and molecular biology techniques. The shattering character relies on the presence of a single-nucleotide polymorphism (SNP) which can explain about 70% of the non-shattering character associated to this gene whereby this SNP probably affects a regulatory region. In my experimental results, the CDS of the eight biotypes of Oryza sativa var. silvatica coincides with a rice genome presenting a high non- shattering character, except for the case of the qSH1-SNP, located outside the CDS, coinciding with the low non-shattering genome rice. In the third year, in order to ascertain if the shattering gene is displaced, breaks or remains on chromosome 1, we made use of the FISH method. The FISH cytogenetic method is employed in order to identify specific sequences of nucleic acids in the chromosome. It represents a significant contribution to standard cytogenetic for the identification of chromosome numerical and structural anomalies. It led however to an encouraging result for two different biotypes: the discovery that the shattering gene does not undergo translocation. Other QTLs for shattering character were noticed on chromosomes 1, 3, 4, 7, 8 and 11, in other cultivated rice species which partially explain the shattering character, although the responsible gene was identified only in three of these QTLs: 1, 4 and 7. The analysis of the involved genes of chromosomes 4 and 7 in our eight red rice biotypes has revealed that the QTLs found on these chromosomes are probably not involved in the shattering character. These analysis provide a wide overview of a possible correlation between the different phenotypes of the eight biotypes, shattering level, karyotypic differences connected with a specific translocation for each biotype and the fact that both these translocations and the gene responsible for the shattering of the eight biotypes are always present in chromosome 1. The investigation of the relationship between the various translocations and the expression of the shattering gene would require further and more sophisticated analyses.